Metabolism and Functions of Glutathione in Micro-organisms

MICHEL J. PENNINCKX" and MARC T. ELSKENS'

a Unite' de Physiologie et Ecologie Microbiennes, Faculte' des Sciences, Universite' libre de Bruxelles, Instut Pasteur Rrabant, 642 rue Engeland, B-I 180-Brussels, Belgium, and (present address) Laborutorium voor Analytische Scheikunde en

Geochemie, Vrije Universiteit Rrussel, Pleinlaan 2, B-I050 Brussels, Belgium

I. Introduction . . . . . . . . . . . . . . . Occurrence and distribution of glutathione and related compounds in micro- organisms , , . . . . . . . . . . . . . .

111. General outlines of glutathione metabolism in micro-organisms . . . A . Biosynthesis: y-glutamylcystcine synthetase and glutathione syn-

thet ase . . . . . . . . . . . . . . B. Degradation:y-glutamyltranspeptidase . . . . . . C. Regulation of the y-glutamyl cyclc . . . . . . . . D. Glutathione metabolism mutants . . . . . . . . . E. Physiological roles of y-glutamyltranspeptidase . . . . . .

IV. Interconvcrsion of glutathione and glutathionc disulphide . . . . . A . Glutathione transhydrogcnases . . . . . . . . . B. The glutaredoxin system . . . . . . . . . . . C. Glutathione peroxidase and the antioxidant defence system in micro-

11.

organisms , . . . . . , , , ,

D. The glutathione redoc cycle . . . . . . V. Conjugation of glutathione: glutathionc Stransferases .

A . Occurrence and distribution in micro-organisms . . B. Substrates and physiological functions . . . .

VI. Otheraspectsofglutathionefunction . . . . . A . The glyoxalase pathway . . . . . . . B. Methanol dissimilation . . . . , . . C. Heavy-metal detoxification . . , , , ,

VII. Concluding remarks . . . . . . . . . VIII. Acknowledgements. . . . . . . . . .

References . . . . . . .

Vides yuum honesti, quani severi dies

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240 M J PENNINCKX A N D M T F I S K F N S

I. Introduction

A century ago, the French scientist de Rey-Pahlade (1888) observed that an ethanolic extract of brewer’s yeast reacted with elemental sulphur to provide hydrogen sulphide. This amazing property was attributed to the presence of a “sulphur-loving” compound first called philothion. The compound was later isolated from yeast and renamed glutathione (GSH) by the English biochemist Frederick Gowland Hopkins (1921). The structure of GSH was established as a tripeptide, y -glutamylcysteinylglycine (y-Glu-Cys-Gly), by chemical analysis, acid-base titration, degradation and synthesis (Kendall et al., 1929; Price and Pinhey, 1929; Harington and Mead, 1935). Since then, it has been demonstrated that GSH is present in high concentration in most living cells from micro-organisms to man. The elucidation of GSH metabolism and its physiological significance in cells has slowly evolved by studies on a variety of biological systems and biochemical reactions. The accelerating rate of data collection on the physiological functions of GSH is reflected by the frequency of symposia regarding this peptide (Colowick et al., 1954; Crook, 1959; FlohC et al., 1974; Arias and Jacoby, 1976; Elliott and Whelan, 1981; Cohen and Friedman, 1982; Larsson et al., 1983; Monks et al., 1990).

The biological importance of GSH is mainly related to the free sulphydryl moiety of the cysteine residue which confers unique redox (E’” = -0.24 V for thiol-disulphide exchange) and nucleophilic properties on the tripeptide. The biosynthesis of GSH is remarkable in two ways: it is mRNA- independent, and the glutamic residue is joined in an unusual peptide linkage of the y -carbon atom to the cysteine residue. Due to this structural peculiarity, GSH is protected against proteolytic cleavage. It follows that a variety of functions have been attributed to GSH.

Obviously, GSH research has ever become more specialized. A computer research of the Index Medicus indicates that more than 10,000 papers on, or quoting, GSH have appeared in the period since the publication of the Fifth Karolinska Institute Nobel Conference on the functions of this compound (Larson et al . , 1983) till mid 1991. Most of them deal with studies on animal cells and, consequently, cover multidisciplinary fields, such as biochemical, physiological, toxicological and clinical aspects. In com- parison, fewer studies were devoted to GSH in micro-organisms or plants (see, however, Penninckx and Jaspers, 1982; Rennenberg, 1982). Neverthe- less, as a result of exchange of ideas, many aspects of GSH metabolism and its functions demonstrated or claimed in animal tissues were also found to apply to micro-organisms. However, substantial differences exist and it is the aim of the following sections to give an up-to-date picture of the development of knowledge on GSH metabolism in prokaryotes and

GI UTATHIONF IN MICRO ORGANISMS 241

microbial eukaryotes. We hope that assessment of the facts given may foster new endeavours and inform microbiologists as to the multiple aspects of this fascinating molecule. We discuss briefly the biologically relevant chemistry of GSH and its occurrence in microbial cells. The GSH-related biochemical reactions and the (possible) physiological roles of GSH are summarized.

11. Occurrence and Distribution of Glutathione and Related Compounds in Micro-organisms

As pointed out by Kosower and Kosower (1978), the GSH status of cells is defined by the total concentration of GSH and the nature and distribution of the possible forms in which the tripeptide can occur in the cell. The most important forms of this compound include reduced GSH, oxidized GSSG and mixed disulphides, mostly GSS-protein or GSSR (R represents a suitable residue such as cysteine or CoASH). Other possibilities are thiol esters which function as intermediates in metabolism of certain compounds such as methylglyoxal and formaldehyde (see Section V1.A). In addition, cellular compounds which behave chemically like GSH or GSSG, such as cysteine, y-glutamylcysteine and reactive disulphides, or which are produced by transpeptidation reactions (see Section 1II.B) like y-glutamyl- peptides, should also be considered in assessing the GSH status of the cell.

Glutathione has long been thought to be the principal low-molecular- weight thiol in many biological systems, but the experimental basis for this generalization has been rather weak, owing to limitations in the available analytical methods. A systematic screening of the occurrence of GSH and related compounds in micro-organisms started at the end of the 1970s with the introduction of a powerful technique based upon the use of bromobimanes, fluorescent labelling agents developed by Kosower and his coworkers (1978, 1979, 1983). Glutathione and soluble non-protein thiol contents were examined by Fahey and his coworkers in a broad spectrum of micro-organisms. In bacteria, hydrogen sulphide was found in all species and was a major compound of many species. The general occurrence of sulphide in bacteria is not surprising and most likely originates from iron- sulphur proteins rather than free hydrogen sulphide (Fahey and Newton, 1983). Glutathione appeared to occur primarily in facultative and aerobic Gram-negative bacteria, but not in strict anaerobes (Fahey et al., 1978). Thiol analysis of bacteria lacking GSH has indicated that CoASH was a major thiol in a number of species, both in Gram-negative and Gram- positive bacteria (Fahey and Newton, 1983) whereas y-glutamylcysteine

242 M J PFNNINCKX AND M T ELSKENF

appeared mainly in halobacteria (Newton and Javor, 198.5). Glutathione, on the contrary, was found to be the major low-molecular-weight thiol in many microbial eukaryotes, including, fungi, protozoa and algae (Fahey and Newton, 1983; Fahey et a f . , 1987; Fairlamb, 1990).

The fact that GSH occurred primarily in organisms with an aerobic life- style suggests that GSH metabolism might have evolved during or after the development of oxygenic photosynthesis. The finding that many bacteria, especially anaerobes, do not produce GSH and that a wide range of phototrophic organisms, purple bacteria, cyanobacteria and eukaryote algae are able to synthesize GSH seems to be consistent with this view (Fahey et a f . , 1987). The only phototrophic bacteria that tolerate oxygen and lack GSH are halobacteria (Newton and Javor, 1985). However, these bacteria, members of the archaebacteria lineage (Fox et a f . , 1980), produce y-glutamylcysteine in large amounts and have a disulphide reductase that maintains it in reduced state (Newton and Javor, 1985). Green bacteria are another group where occurrence of GSH is questionable, but most green bacteria are obligate anaerobes carrying out anoxic photosynthesis. So, it has been suggested that endosymbiotic processes giving rise to mitochondria and chloroplasts might represent a plausible mechanism for acquisition of GSH synthesis in eukaryotes (Fahey et al., 1987). Entomoeba histolytica, which lacks both chloroplasts and mitochondria, is indeed the only eukaryote that has been clearly demonstrated not to produce GSH (Fahey et a f . , 1984).

The radioprotective effect of GSH as well as the correlations found between radiosensitivity variations of cells and their GSH contents were put forward as an argument for the concept that the tripeptide can be an intrinsic cellular radioprotector of special importance (Fuchs and Warner, 1975; Kosower and Kosower, 1978; Revesz and Malaise, 1983). It is therefore tempting to postulate that the initial function of GSH, when oxygen became a significant component of the atmosphere, was protection of cells against oxygen toxicity by destruction of thiol-reactive oxygen by- products (Fahey el a f . , 1987). However, there is little other evidence to support this view. The finding that many bacteria, including some strict aerobes, lack GSH but contain other thiols suggests that more than one thiol-based protection system could have evolved in prokaryotes. Moreover, the apparent absence or virtual absence of GSH transferase and GSH peroxidase, some of the key enzymes involved in oxygen detoxification (see Sections 1V.B and V) in Escherichia coli (Smith and Shrift, 1978; Lau et al., 1980) and in Saccharomyces cerevisiae (Smith and Shrift, 1978; Aisaka et al., 1983), raises serious questions about the role of GSH in these organisms. It is possible, therefore, that GSH played entirely different functions in early bacteria, and that the oxygen detoxification

GLU~IAI'I IIONE IN MICRO-ORGANISMS 243

C/"\ ,CH3

kC-N I y,/ c\ CH,

0 'c C-OH \

d

H I

P Y HO-C -C

I NHZ

C-N H / o* ' L

H'

(b)

FIG. 1. Comparison of the structurc of (a) isopcnicillin N and (b) the p-lactam form of GSH (glutacillin). From Spallholz (1987).

function evolved only later. In this connection, the hypothesis of Spallholz (1987) should be considered. Glutathione is structurally similar to the precursor of the antibiotics produced in fungi in the genera Penicillium and Cephalosporiurn. Its potential conversion to the penicillin-like derivative glutacillin, a p-lactam form of GSH, raises the intriguing question whether glutathione was once a universal penem-like precursor of antibiotics in cells of many life forms (Fig. 1). The loss of the ability to convert GSH (if it ever existed) is, of course, open to speculation. It should be noted that the emergence of cellular immune systems with an apparent role for GSH and specific phagocytosis in higher organisms may have evolutionarily displaced the need for formation of natural antibiotics in higher organisms. The oral activity of glutathione against post-tumour induction in rats was found to be common with the oral activity of many penicillin derivatives against bacteria (Spallholz, 1987).

The intracellular content of GSH is variable according to its distribu- tion and occurrence in micro-organisms. Under normal, unstressed

244 M 1 PENNlNCKX AND M 7 kLSKENS

physiological conditions, much of the tripeptide is present in the free reduced form. In Sacch. cerevisiae (Penninckx et al., 1980) and E. coli (Newton and Fahey, 1990), the GSH content is very high and accounts for more than 1% of cell dry weight. The concentration of the oxidized form, GSSG, is usually much smaller, with reported values for the GSH-GSSG ratio generally being greater than 50. The balance between the thiol and disulphide groups is essentially maintained by the widely distributed GSH reductase (see Section 1V.D) ensuring a cellular environment in which essential sulphydryl groups of key enzymes and co-enzymes are protected. Modest changes in the rather low concentration of GSSG may be critical for regulation of certain physiological processes (Kosower and Kosower, 1978). Refined analytical methods are now available for very sensitive determinations of GSH and GSSG pools (Meister, 1985; Eyer and Podhradsky, 1986; Fahey and Newton, 1986) and considerable efforts have been undertaken to optimize extraction procedures in several kinds of organisms (Fahey and Newton, 1983; Fahey et al., 1987).

Mixed disulphides have not been studied as extensively in micro- organisms (see, however, Fahey et al., 1975). The mixed disulphide GSS- protein represents, in most organisms, intermediate forms with enzymes. These associations could reflect either a regulation of enzyme activity as with inorganic pyrophosphatase in Steptoccoccus faecalis (Lahti and Suonpaa, 1982) or modulation of protein conformation by thiol-disulphide exchange reactions (Pryor, 1962; Freedman and Hillson, 1980). It has been shown that the mixed disulphide CoASSG is a major component of the CoA pool in yeast (Stadtman and Kornberg, 1953) and E. coli (Loewen, 1981). The disulphide inhibits RNA polymerase and its reduction is catalysed by a specific enzyme in E. coli. The pool of CoA does not change much in mutants affected in GSH biosynthesis, but strains deficient in y- glutamylcysteine synthetase (gshA) produce only the CoA dimer whereas mutants impaired in GSH synthetase (gshB) produce the mixed disulphide of CoA and y-Glu-Cys (Loewen, 1981).

Another interesting and important derivative is the covalent adduct GSH- spermidine formed at the end of exponential growth by E . coli (Tabor and Tabor, 1979) and in trypanosomatids (Fairlamb et al., 1985, 1986). In E coli, the product probably undergoes a rapid turnover and, therefore, may exist at a very low steady-state level in exponentially growing cells. Two specific enzymes, catalysing synthesis of the product from spermidine, GSH, ATP and magnesium ions and its hydrolytic degradation were, respectively, present during the entire growth stage. Glutathionylspermidine may play a role in regulation of growth and nucleic acid metabolism (Tabor and Tabor, 1975). In trypanosomes and leishmania, about 80% of GSH is present as N'-glutathionylspermidine and N' , fl-bis(glutathiony1)

GLUl A r H l O N t I N MICRO-ORGANISM\ 245

spermidine; the latter compound is unique to trypanosomatids and was once called trypanothione (Fairlamb et al., 1985). The biosynthetic pathway to trypanothione has been established by radiolabelling and inhibitor studies (Fairlamb et al., 1986, 1987; Bellofato et al . , 1987). A single enzyme catalysing both N’-mono-, N8-monoglutathionylspermidine and trypano- thione biosynthesis from ATP-Mg2+, GSH and spermidine has been purified approximately 14,500-fold to homogeneity in an overall yield of 40% (Henderson et al., 1990). The enzyme was active in monomeric form ( M r 87,000) and has a turnover number of 1700 min-’ with GSH and spermidine. It has been suggested that trypanothione has important physiological functions in trypanosomatids. In the first place, due to the absence of glutathione reductase (Fairlamb and Cerami, 1985), GSSG is reduced non-cnzymically by thiol-disulphide exchange with hydro- trypanothione (T[SH],). The resulting cyclic trypanothione disulphide (TS,) is reduced in turn by an NADPH-dependent flavoenzyme, trypanothione reductase (Shames et al., 1986; Jockers-Sherubl et al., 1989). Secondly, a trypanothione peroxidase activity that could contribute to protection of cells against oxygen damage was identified in Trypanosoma brucei and Crithidia fasciculata (Henderson et al., 1987; see also Section 1V.B). The importance of the trypanothione system is also of considerable interest in the development of chemotherapy against tropical diseases caused by parasitic trypanosomes (African sleeping sickness and Chagas’ disease) and leishmania (cutaneous and visceral leishmaniasis). A number of existing drugs have already been shown to interact with this important area of~metabolism (see Fairlamb, 1989, 1990).

Amohg the different forms of GSH-related compounds are the peptides (y-Glu-Cys),-Gly produced in the presence of cadmium salts by Schizosaccharomyces pombe (Grill et al, 1985) and Candida glabrata (Mehra et al., 1988). These compounds are presumably involved in heavy- metal detoxification (see Section V1.C). In addition, several low-molecular- weight y-glutamyl compounds, including dipeptides and more complex mglCcules, were shown to be produced by micro-organisms. Little is known about their physiological roles (see, however, Section 1II.E) and their link with GSH metabolism is not always demonstrated. While there is some evidence that synthesis of y-glutamylpeptides occurs in vivo by transpeptida- tion reactions in Sacch. cerevisiae after growth on glutamate as the nitrogen source (Jaspers et al., 1985), and in Corynebacterium glutamicum, during the L-glutamic acid fermentation (Hasegawa and Matsubana, 1978), y- glutamyl compounds are also produced by other pathways lacking direct relationship with GSH metabolism. For example, in the koji mold Aspergillus oryzae (Tomita et al., 1989) and Bacillus natto (Noda et al., 1980), a glutaminase (or transamidase) catalysed formation of y-glutamyl compounds

2,46 M J PFNNINCKX AND M T FISKFNS

from glutamine and amino acids. Several analogues of GSH were also reported. Ophthalmic acid (y-L-glutamyl-L-a-aminobutyrylglycine) and norophthalmic acid (y-L-glutamyl-L-alanylglycine) were isolated from the algae Undaria pinnatijida (Ogawa et al . , 1990). The precursors of oph- thalmic acid, a-aminobutyrate and y-glutamyl-r,-a-aminobutyrate, were often substituted to cysteine and y-glutamylcysteine, respectively, in the in vitro assays of y-glutamylcysteine synthetase (y-GCS) and GSH synthetase activities (Mooz and Meister, 1967). N-(N-y-Glutamyl-3-sulpho- L-alany1)glycine was found in the mushroom Flammulina vetupilis (Ogawa et al., 1987) and l-y-glutamyl-2-(2-carboxyphenyl)hydrazine is produced by Penicillium oxalicum (Minato, 1979). The latter compound, trivially named anthglutin, was also synthesized as the result of a rational chemical design for y-glutamyltranspeptidase (y-GT) inhibitors (Griffith and Meister, 1979a).

y-Glutamyl- - Amino-acid transpeptidase transport

integrity

FIG. 2. Diagrammatic representation of the intcrrelationship of GSH with other cellular biochemical systems (Mitchell, 1988).

GI UTATHIONF IN MICRO ORGANISMS 247

The present data are a good illustration of the diversity regarding occurrence of GSH and related compounds in the microbial world, and provide some insight into the complexity of thiol biochemistry in micro- organisms. A precise expression of GSH status, as proposed by Kosower and Kosower (1978), involving measurements of GSH contents, a quantita- tive description of the different chemical forms of the tripeptide and related compounds and an assessment of their spatiotemporal variations within the cell, is still difficult to establish for micro-organisms due to a lack of knowledge of certain reactions. Nevertheless, one should bear in mind that the term “status” does not imply a fixed or constant value for GSH contents, but refers rather to a description of a dynamic system with a shifting of the equilibrium among different forms in response to natural or artificial perturbations. Although GSH appears not to be essential in prokaryotes, its uniform distribution in eukaryotes suggests that it might serve essential functions. Amid the complex machinery of cellular biochemistry, the tripeptide assumes a pivotal role in numerous bioreductive reactions, transport, enzyme activity, protection against harmful oxidative species, and detoxification of xenobiotics. Having such functional diversity, GSH is interrelated with a number of biochemical systems (Fig. 2). As for animal cells, GSH-related enzymes in micro-organisms can be grouped into those concerned with biosynthesis, degradation, reduction, oxidation, conjugation and those in which GSH serves as a cofactor.

111. General Outlines of Glutathione Metabolism in Micro-organisms

The scheme given in Fig. 3 outlines the biochemistry of GSH and associated pathways that were identified or claimed to exist in micro-organisms. Many investigators in this field were largely influenced by studies on animal tissues and tried firstly to identify similar pathways in micro-organisms. Differences appeared in the course of these investigations, and some major features regarding enzymes and biochemical phenomena involved have emerged only quite recently.

Several lines of evidence indicate that GSH metabolism proceeds in higher eukaryotes through the y-glutamyl cycle (Meister, 1981a; Rennenberg, 1982; Meister and Anderson, 1983). In its original version, the cycle accounts for six reactions whose three steps are ATP-dependent, namely the two consecutive reactions of GSH biosynthesis (equations (1) and (2)) and conversion of 5-oxo-~-proline to i,-glutamate (equation (6)). Modifications of the y-glutamyl cycle have been discussed in detail elsewhere (Meister, 1981b). y-Glutamyltranspeptidase, following hydro- lysis and transpeptidation reactions, provides an alternative pathway in

248 M. J PENNINCKX AND M. 7. ELSKENS

\ / \ GLY \

5-Oxoproline

\ ATP

FIG. 3. The y-glutamyl cycle (Meister, 1983). The enzymes involved are (1) y- Glutamylcysteine synthetase, (2) glutathione synthetase, (3) y-glutamyltranspeptidase, (4) cysteinylglycine dipeptidase, (5) y-glutamylcyclotransferase and (6) 5-oxoprolinase.

which activities of y-glutamylcyclotransferase and 5-oxoprolinase are excluded. Until now, only little evidence has been presented for the existence of a complete y-glutamyl cycle in micro-organisms (Jaspers et al., 1985), even though a first report for baker's yeast was published in 1976 (Mooz and Wigglesworth, 1976). In strains of Saccharomyces cerevisiae, GSH catabolism appears to be mediated by y-GT and cysteinylglycine dipeptidase only, and it was observed that the latter activity is shared by several peptidases constitutively produced by this organism (Jaspers et al., 1985). Both y-glutamylcyclotransferase and 5-oxoprolinase activities were undetected in crushed or permeabilized yeast cells. Direct labelling experi- ments have shown that ['4C]5-oxoproline (pyroglutamic acid) was taken up intact, but not further metabolized into glutamate (tllz being 1000 minutes).

y-Glutamyltranspeptidase was described long ago in many bacteria (Milbauer and Grossowicz, 1965) and was even suggested as a useful indicator for identification of members of the Enterobacteriaceae (Giammanco et al., 1980). A dipeptidase was also shown in Bacillus cereus (Cheng et al., 1973). More recently, a typical 5-oxoprolinase from a Pseudomonas putidu strain was purified and characterized (Li et ul., 1988). As far as is known, this remains an isolated but substantiated observation in the

GLUTATHIONE IN MICRO-ORGANISMS 249

microbial world. Hence, the current picture emerging from these investiga- tions into micro-organisms suggests that, for most of them, a truncated version of the y-glutamyl cycle, involving the biosynthesis enzymes y-GT and cysteinylglycine dipeptidase, would exist. Quite apart from this debate, it is clear that the cycle concept introduced by Meister as a result of observations on animal tissues has been very useful as a working hypothesis for a number of investigations on the biochemical function of GSH. This concept has led to many findings about glutathione and has radically altered the understanding of metabolism of the thiol tripeptide in living cells.

A. BIOSYNTHESIS: y-GLUTAMYLCYSTEINE SYNTHETASE AND

GLUTATHIONE SYNTHETASE

Glutathione is synthesized intracellularly by the consecutive action of y-GCS (L-y-glutamate-L-cysteine y-ligase (ADP); EN 6.3.2.3) and GSH synthetase (L-y-glutamy1cysteine)-glycine y-ligase (ADP); EN 6.3.2.3) (Snoke and Bloch, 1954). Both enzymes require ATP and magnesium ions for activity. There is some evidence that enzyme-bound y-glutamylphosphate and y- glutamylcysteinylphosphate are formed in these reactions, whose mechanisms are thus similar to those catalysed by glutamine synthetase (Meister 1983). y-Glutamylcysteine synthetase (y-GCS) has been isolated from several sources, but only recently purified and characterized from a microbial source (Watanabe et al., 1986). The enzyme from Escherichia coli consists of a single polypeptide chain ( M , 55,000) differing from the rat-kidney enzyme (Orlowski and Meister, 1971), which dissociated into two non-identical subunits ( M , 74,000 and 24,000) and from the enzyme from Proteus mirabilis, which separated into three subunits with respective molecular weights of 30,000, 11,000 and 13,000 on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis (SDS-PAGE) (Kumagai et al. , 1982). However, the possible presence of proteolytic contaminants was apparently not checked in the latter report. The complete nucleotide sequence of the gene coding for y-GCS in E. coli has been reported (Watanabe et al., 1986). The polypeptide deduced from the open-reading frame has a molecular weight that agrees with values+reviously determined by SDS-PAGE and gel filtration (Murata and Kimura, 1990). As already shown for the rat-kidney enzyme (Richman and Meister, 1975) the bacterial enzyme is also inhibited by physiological concentrations of GSH with a Ki value of 2.5 mM (Murata and Kimura, 1990). Presumably, this indicates a physiologically significant feedback mechanism. Molecular cloning of the y-GCS gene from Sacch. cerevisiae has shown that the GSHA gene comprises a segment of 2034 bp that encodes for a protein with about 678

250 M J PENNINCKX A N D M T b L S K b N S

amino-acid residues (Ohtake and Yabuuchi, 1991). The deduced amino- acid sequence presented 45% homology with the rat-kidney enzyme (Yan and Meister, 1990), but only 26% with the enzyme from E. coli (Watanabe et al., 1986).

L-Methionine (S)-sulphoximine is an inhibitory analogue of the enzyme- bound y-glutamylphosphate intermediates formed in reactions catalysed by both glutamine synthetase and y-GCS (Meister, 1983). The rational design and synthesis of new analogues which produce selective inhibition of these enzymes have been developed by Meister and his coworkers (Griffith et al., 1979; Griffith and Meister, 1979b). Buthionine (S,R)-sulphoximine (BSO) is an analogue inhibitor of the transition state of y-GCS-bound substrates (y-glutamylphosphate and cysteine) and is at least 200 times more active than methionine sulphoximine. Since the S-butyl moiety of BSO prevents its interaction with glutamine synthetase, the new inhibitor is very selective. Depletion of glutathione by BSO has proved to be a very useful method for decreasing GSH levels in many organisms (Meister, 1988) and has several advantages over the use of oxidizing agents and compounds that react with GSH itself.

The second enzyme, GSH synthetase, was purified from yeast 25 years ago (Mooz and Meister, 1967). An apparent molecular weight of 123,000 was first deduced from ultracentrifugation experiments. Further investigations using gel filtration and SDS-PAGE indicated molecular weights of, respectively, 152,000, 147,000 and 152,000 for enzymes from yeast (Meister, 1974), P. mirabilis (Nakayama, 1984) and E. coli (Gushima et al., 1983). The purified enzyme from E. coli is apparently composed of four identical subunits ( M , 38,000). Crystallization and preliminary X-ray studies were recently reported. Rather amazingly, a strong homology between GSH synthetase from E. coli and mammalian and bacterial dihydrofolate reductases was shown over 40 amino-acid residues, in spite of the fact that these enzymes differ in their reaction mechanisms and ligand requirements (Kato et al., 1987). Some current studies are following the trend of elucidating evolutionary pathways of these enzymes (Murata and Kimura, 1990).

B . DEGRADATION: y-GLUTAMYLTRANSPEPTIDASE

y-Glutamyltranspeptidase (y-GT; E C 2.3.2.2.) is an enzyme of major importance in GSH metabolism although its physiological role is not yet fully understood. As previously stated, y-GT is widely distributed in bacteria and has also quite recently been isolated from Mycobacterium smegmatis, a representative species of the actinomycetes (Kumar et al., 1990). In microbial eukaryotes, the enzyme was detected in yeast (Mooz

GLUTATHIONF IN MICRO-ORGANISMS 25 1

and Wigglesworth, 1976; Penninckx et al., 1980), in the moulds Tricholoma shimeji (Iwami et al., 1978) and Aspergillus oryzae (Tomita et al., 1988), and in the epimastigotes of the protozoan Trypanosoma cruzi (Repetto et al., 1987).

In E. coli and P. mirabilis, y-GT is localized in the cell walls and periplasmic space or both, as shown by studies with lysozyme-EDTA and fluorescent antibodies (Nakayama et al., 1984; Suzuki et al., 1986). In Sacch. cerevisiae, the enzyme appears as a membrane-bound entity localized mainly in vacuoles (Jaspers and Penninckx, 1984) and/or in the plasma- lemma (Payne and Payne, 1984). Fully cytosolic forms were reported for T. cruzi and M . smegmatis (Repetto et al., 1987; Kumar et al., 1990). An excreted form was also detected in A. oryzae (Tomita et al., 1988). The enzyme was extensively purified from P. mirabilis (Nakayama et al., 1984), E. coli (Suzuki et al., 1986) and from Sacch. cerevisiae (Jaspers and Penninckx, 1985). In all instances, the enzyme was dissociated into two different subunits as previously shown for the mammalian form. However, in mammals, y-GT appears to be a glycoprotein and this was only once reported for the enzyme from Sacch. cerevisiae. The DNA sequence of the gene in E. coli has been determined recently (Suzuki et al., 1989). The sequence contained a single open-reading frame, encoding the signal peptide and both subunits, which suggests a post-translational processing of y-GT.

Purified microbial y-GTs can catalyse, as the mammalian enzyme, three types of reactions: (a) hydrolysis in which the y-glutamyl moiety is transferred to water; (b) transpeptidation in which the y-glutamyl moiety is transferred to an amino-acid or peptide acceptor; and (c) autotrans- peptidation in which the y-glutamyl moiety is transferred to GSH. Glutathione (GSH and GSSG), S-substituted GSH and numerous y- glutamyl compounds are potential substrates for y-GT. L-Cystine, methionine and glutamine are among the most active acceptors, but other amino acids, as well as many dipeptides, especially aminoacylglycines, also participate significantly in transpeptidation (Meister and Anderson, 1983; Penninckx and Jaspers, 1985).

y-Glutamyltranspeptidase is inhibited specifically and competitively by L- and D-isomers of y-glutamyl(o-carb0xy)phenylhydrazine and related compounds (Griffith and Meister, 1979a; Minato, 1979). A combination of L-serine and borate apparently inhibits y-GT by forming an analogue of the transition state (Tate and Meister, 1978). Several glutamine analogues, such as L-azaserine, 5-oxo-~-norleucine and ~-[aS,5S]-u-amino-3-chloro- 4,5-dihydro-5-isoxazole acetic acid were also described as non-specific but potent irreversible inhibitors of y-GT (Tate and Meister, 1978; Allen et al., 1980; Griffith and Meister, 1980).

252 M. J. PENNIN('KX AND M. 7 F I S K E N S

C. REGUCATION OF T H E y-GLUTAMYL CYCLE

The intracellular concentration of GSH reflects the equilibrium between biosynthesis and degradation. In Sacch. cerevisiae, y-GT synthesis was found to be regulated by at least two apparently distinct pathways (Table 1). In the presence of ammonium ions as a nitrogen source, the transpeptidase level is low in the wild-type strain (about 40 nmol h-l (mg protein)-'). In the presence of glutamine, arginine, ornithine or citrulline, the enzyme level rises to an intermediate value (8&100 nmol h-' (mg protein)-') and the highest specific activities are observed with proline, urea or glutamate (200-225 nmol h-' (mg protein-'). When cells of Sacch. cerevisiae are starved for two hours by transfer to a medium devoid of a nitrogen source, the level of cellular enzyme rises to the highest value (200 nmol h-' (mg protein)-'). A study of the rate of y-GT synthesis showed that, after transfer from a proline-supplemented medium to a medium containing ammonium

TABLE 1 . Regulation of the y-glutamyltranspeptidasc in Sacchuromyces cerevisiae. The enzyme specific activity (as rate of release of p-nitroaniline; see Penninckx et al., 1980) was estimated in crude extracts from exponential-phase cells growing on the nitrogen sources

listed in thc table ~

Strains and genotypes Nitrogenous nutrients Enzyme specific activity (nmol h-' (mg protein)-')

-~

Z1278b (wild type) Ammonium ions Proline Urea

gshA

g d h A

gdhCR

Glutamate Ammonium ions Ammonium ions with GSH Glutamate Glutamate with CSH Ammonium ions Glutamate Urea Ammonium ions Glutamatc Urea

<40 200 225 225 98 60

430 270 45

225 240 50

210 235

gap - Ammonium ions <40 Proline 200

apf Ammonium ions <40 Urea t40

Urea 240

Urea 260 argp-, lysp- Ammonium ions < 40

Urea 240

argp - Ammonium ions <40

argp -. gap-. Ammonium ions <40

GI UTATIIIONF IN MICRO-ORGANISMS 253

ions, the enzyme is expressed at the low rate characteristic of the minimal medium. This result is consistent with a repression mediated by ammonium ions (Pennickx et al., 1980). The repressive effect of ammonium ions on the transpeptidase was found to be unaffected in mutants (gdhA, gdhCR) which have lost the regulatory circuits governed by glutamine and the glutamate dehydrogenase-NH4" complex (Table 1 ) . Taking into account that most of the nitrogen metabolic pathways in Sacch. cerevisiae were found to respond to at least one of the two above-mentioned signals (Dubois et al., 1977), it seems that regulation of the transpeptidase level depends on a different mechanism.

The second regulation mechanism was discovered in the course of an investigation into the biochemical characterization of a GSH-deficient yeast mutant (gshA) impaired in the first step of glutathione biosynthesis. As shown in Table 1, GSH deficiency has a noticeable influence on the specific activity of y-GT. In the Gsh- phenotype, the specific activity of y-GT was about twice (9C100 nmol h-' (mg protein)-') that in the wild-type strain growing on the same ammonia-containing medium. Addition of GSH to a culture of a deficient strain decreased the differential rate of y-G1' synthesis to a value close to that of the wild type. In contrast, derepression of y-GT synthesis was observed when the GSH level in the wild-type strain was lowered by the use of BSO. It is interesting to note that the rate of y-GT synthesis increases drastically when the intracellular concentration of GSH falls below 4 nmol (mg dry weight)-'. The enzyme derepression which appears constitutive in gshA mutants results, therefore, most likely from an alteration of the thiol status and correlates best with a decrease in the GSH pool among the sulphur-containing compounds examined (Elskens et al., 1991). The repression mediated by ammonium ions is still present in GSH- deficient strains and the highest specific activity is again observed with glutamate, proline or urea as the sole nitrogen source (Table 1). Promotion of the y-GT synthesis caused by GSH deficiency and the absence of a better nitrogen source results from a simple cumulative effect. The extent of derepression is relatively weak, with a 2-10-fold increase in activity when both effects are combined.

Derepression of y-GT synthesis in Sacch. cerevisiae is correlated with a strong increase in GSH turnover rate. From a value of about 900 minutes on a minimal medium containing ammonium ions the half-life of intra- cellular GSH is decreased to approximately 200 minutes with glutamate as the only nitrogen source (Jaspers et al., 1985). Since the increase of turnover rate does not lower the intracellular GSH content under normal unstressed physiological conditions (Jaspers and Penninckx, 1981; Robins and Davies, 1981b; Jaspers et al., 1985), a net increase in the GSH biosynthesis rate is expected.

254 M J PFNNINCKX AND M 1 kLSKENS

In yeast, the intracellular level of GSH synthetase is apparently unaffected by the nature of the nitrogen source (Jaspers et al., 1985), but y-GCS is feedback inhibited by GSH and is apparently the rate-limiting step in biosynthesis of the tripeptide (Ohtake et al., 1989). Biotechnological investigations dealing with GSH biosynthesis by genetically engineered cells of E. coli support this view (Murata and Kimura, 1990). Molecular breeding experiments with hybrid plasmids having the genes encoding GSHA and GSHB have shown that the first step in glutathione biosynthesis is rate- limiting, and that a simultaneous increase in activities of both the GSHA and GSHB enzymes resulted in a higher production of glutathione. It could be suggested, therefore, that GSH might be able to regulate its own formation and maintain a steady-state equilibrium between synthesis and degradation by acting on y-GCS only (see Kumagai et al., 1982). However, the situation might be more complicated. In Penicilliurn chrysogenum, a stimulatory effect of ammonium ions on GSH formation has been observed (Schwartz et al., 1988). High concentrations of the ion stimulated not only formation of GSH, but also increased the intracellular pools of glutamate, alanine and glutamine. The induction effect of glutamate on GSH synthesis by stimulating y-GCS activity was shown in resting cells of the wild-type strain Pe. chrysogenurn. In yeast, it would not be very surprising to find that the availability of nitrogenous nutrients plays a leading role in determining rates of GSH formation because synthesis of enzymes involved in the amino-acid biosynthetic and catabolic pathways is regulated (Jones and Fink, 1982). Nevertheless, as specific regulatory controls in amino- acid biosyntheses are not common in yeast (Messenguy et al., 1980), the possible inductive effect of glutamate on y-GCS remains to be demonstrated for this organism. The carbon source has been found to affect GSH production in yeast. A recent study, emphasizing optimal production of GSH in which the specific growth rate of Sacch. cerevisiae was controlled in glucose-fed batch culture, has shown that, when the growth rate was kept at sufficiently low values, ethanol was not produced and the intracellular level of GSH was about 2540% higher than under conditions where ethanol is produced (Shimizu et al., 1991). It is predictable, therefore, that GSH biosynthesis may be under the influence of the so-called glucose effect (Pasteur and Crabtree effects) which is observed in a larger number of yeast species. One should stress, however, that glucose regulation, especially in yeasts, includes an extensive number of enzymes and is probably based on a rather complicated regulatory situation in terms of underlying mechanisms. It seems, indeed, that catabolite repression represents only one of the mechanisms involved, and it could be accom- panied by other regulatory mechanisms, specific controls of protein synthesis and modification as well as catalytic turnover of enzymes (Fiechter

CLU TATHIONE IN MICRO-ORGANISMS 255

et al., 1981). So far, it would appear that the effect of glucose limitation on GSH biosynthesis may be direct or indirect and needs to be investigated in greater detail before the question is definitively answered.

Besides the effects of nitrogenous and carbon nutrients, perturbation of the intracellular GSH status has been reported in relation to the cell cycle, growth and development. During the exponential growth phase of Sacch. cerevisiae, the intracellular concentration of GSH remains almost constant, but increases drastically in a reproducible way within fairly narrow ranges when measured under the same conditions at the beginning of the stationary phase of growth. Changes in the intracellular GSH content during induction of the yeast-to-mycelial transition in Candida albicans was also reported. As emphasized by Kosower and Kosower (1978), variations, both in time and space, are natural for the GSH status of cells. Serious deviations from usual or normal values can occur as a consequence of (a) normal physiological situations, (b) genetic defects, and (c) chemical reactions resulting from addition of exogenous agents. In the last case, most of the perturbations decrease the GSH content by chemical reactions and biochemical processes, such as oxidation and conjugation. Given the current GSH status for a cell, a perturbation can be either reversible or irreversible. Reversible situations mostly involve oxidation of the reduced form into GSSG and mixed disulphides, but also include acylation of GSH. Irreversible perturbations take place when GSH synthesis fails, if GSH is converted into covalently bound derivatives or when GSH or GSSG is eliminated from the cell. Unlike animal cells, GSH and its derivatives are not normally excreted into the surrounding culture medium by whole micro- organisms, but leakage was reported for some strains of Sacch. cerevisiae growing in the presence of selenite or ethanol (Izuka et al., 1988) and in filamentous forms of Candida tropicalis (Yamada et al., 1984). Similarly, inhibition of y-GT by serine-borate causes leakage of GSH from whole P. mirabilis (Nakayama et al., 1984) and in some mutants of E. coli (Murata et al., 1980).

D . GLUTATHIONE METABOLISM MUTANTS

y-Glutamylcysteine synthetase (gshA)- and GSH synthetase (gshB)- deficient mutant strains have been isolated and characterized in bacteria (Apontoweil and Behrends, 1975b; Fuchs and Warner, 1975; Murata et al., 1981; Fuchs et al . , 1983; Kerklaan et al. , 1985; Bouter et al., 1988) and in yeast (Kistler et al., 1986; Glaeser et al., 1991).

Apontoweil and Behrends (1975a,b) isolated a mutant of E. coli impaired in y-GCS by selecting cells with increased diamide sensitivity. The gshA mutant contained no detectable glutathione in a trichloroacetic acid

256 M J. PENNINCKX AND M. T. EISKENS

extract of [35S]sulphate-labelled cells. Comparative assays for y-GCS indicated that the mutant had only 4% of the parental activity. Although the growth rate of the Gsh- phenotype appeared unaltered by glutathione deficiency, the cells were 3-10-fold more sensitive to sulphydryl reagents, antibiotics, chemotherapeutic agents, food additives, pesticides and other miscellaneous chemicals. The gshA mutation mapped at 57 minutes on the E. coli chromosome (Apontoweil and Berends, 1975b).

Concurrently, with isolation of a y-GCS-defective E. coli mutant, Fuchs and Warner (1975) isolated a gshB mutant in an attempt to obtain thioredoxin reductase mutants (the thioredoxin pathway provides to some extent an alternative pathway for reactions that may otherwise require glutathione; see Section 1V.B). Glutathione was not detected in the gshB mutant, neither when the classical enzymic assay was used for GSH determination nor when the trichloroacetic acid fraction was labelled with ['4C]N-ethylmaleimide, a thiol alkylating reagent. However, the mutant had a significantly elevated pool of y-glutamylcysteine. Assays for GSH synthetase indicated that the mutant had less than 10% of the parental enzyme activity. The gshB mutant grew at the same rate as the parental strain, but its growth was inhibited at a three-fold lower concentration of diamide or methylglyoxal. It was found to have an enhanced sensitivity to X-rays in the absence of oxygen (Morse and Dahl, 1978), but not when oxygen was present (Fuchs and Warner, 1975). The gshB mutation mapped near xyl by Hfr matings (Fuchs et al. , 1983).

To diminish the possibility that a small but significant amount of glutathione might be synthesized in either the gshA and the gshB mutant, Fuchs and his coworkers (1983) constructed a (gshA gshB) double mutant. A TnlO transposon was introduced into a gene required for utilization of sorbitol (srl) by Csonka and Clark (1980). From the mapping data of Apontoweil and Berends (1975b), the gshA gene should cotransduce with this sr1:TnlO with a frequency of about 66%. A sr1:TnlO gshA strain was thus constructed and subsequently used to transduce the gshB mutant to tetracycline resistance. The double mutant did not differ in growth characteristics from the gshB parent, which suggests that GSH is not required by E. coli under laboratory growth conditions (Fuchs et a l . , 1983).

As previously stated, a strain of E. coli enriched in its content of y-GCS (GSHA) and GSH synthetase (GSHB) activities by recombinant DNA techniques was also characterized (Moore et al. , 1989; Murata and Kimura, 1990). The proficient enriched strains had a higher GSH content than their corresponding wild type and appeared much more resistant to the lethal effects of y-irradiation. The radioresistance, however, was shown to correlate better with increased ability of the gene-enriched strain to synthesize GSH rather than to the cellular level of the tripeptide per se. It

CLU TATHIONE IN MICRO ORGANISMS 257

follows that resistance can be abolished by BSO, a specific y-GCS inhibitor that decreases the rate of GSH biosynthesis but does not react directly with the tripeptide (Moore et al., 1989).

Glutathione-deficient mutants (gshA) of Sacch. cerevisiae were isolated after ultraviolet irradiation using the N'-nitro-N-nitrosoguanidines as selective agents (Kistler et al . , 1986). Since the nitrosoguanidines (MNNG or ENNG) are activated by GSH in bacteria, yeast and mammalian cells (Mohn et a f . , 1983), selection was based upon isolation of the Gsh- phenotype as MNNG- and/or ENNG-resistant clones. All isolates were impaired in at least the first step of GSH biosynthesis (gshA). Residual activity of y-GCS was found to be about 5-10% of the parental activity (Kistler et al., 1990). The mutants exhibit considerably lower residual GSH contents, varying from 2 to 8% of the wild-type strain. All isolates show a 2:2 segregation of the Gsh-: GSH' phenotypes, suggesting a monogenic recessive mutation. Complementation analysis furthermore indicated that all mutants belong to one complementation group. Deficiency of GSH leads to pleiotropic phenotypes of the mutants. Lower GSH levels in the haploid Gsh- strains are correlated with an extension of the lag phase of growth and a decrease in growth rate when cells were cultivated on rich or synthetic media (Kistler et al., 1986). Similar conclusions were reached by Elskens et a f . (1991) from experiments with BSO. Cells containing more than about 1 nmol GSH (mg dry weight)-' are apparently able to grow normally. At present, it is unknown if a complete lack of GSH is lethal in Sacch. cerevisiae as the growth rate decreases dramatically below this value. Kistler et al. (1986) reported that a residual GSH level of 1%" of the wild-type value, as found in some gshA mutants, leads to serious impairments in the viability and respiratory ability, accompanied by loss of mitochondria1 DNA.

Prototrophic Gsh- derivatives (for filiation of the strains, see Elskens et al., 1991) show in various degrees, as already described for the original isolates, the pleiotropic effect of the mutation on growth and sexual reproduction. But it was also demonstrated that they presented striking differences in contents of the sulphur-containing amino acids and thiol status when compared with wild-type strains growing on the same minimal medium containing ammonium ions. Similar to gshA mutants of E. coli, GSH-deficient strains of Sacch. cerevisiae were found to be more sensi- tive to sulphydryl reagents such as diazene derivatives (e.g. diamide), dithiocarbamate pesticides and chloro- or fluorodinitrobenzene compounds.

It is important to note that the selection procedure may influence isolation of GSH-deficient mutants. According to Kistler and his colleagues (1990), y- GCS-deficient strains have been much more frequent among selected Gsh- mutants of E. coli. Furthermore, when nitrosoguanidine (MNNG, ENNG) resistance was the selected phenotype, only y-GCS-deficient

25 8 M J PENNINCKX A N D M T ELSKENS

mutants were isolated from Salmonella typhimurium and E. coli (Bouter et al., 1988). There is substantial evidence that GSH-mediated activation of nitrosoguanidines is performed in bacteria by dipeptide y-glutamyl- cysteine as well, especially when it is accumulated as a result of a GSH synthetase deficiency. Therefore, it has been suggested that MNNG selection would probably be inappropriate to detect GSH synthetase- defective mutants (Kistler et al., 1990). It appears, however, that such mutants of Schizosaccharomyces pombe were obtained by this procedure (Glaeser et al., 1991).

Genetic defects related to GSH biosynthesis are considered to be a useful tool in determining some of the roles of GSH in micro-organisms. In spite of the fact that GSH is apparently not essential for growth of E. coli under usual laboratory conditions, it is likely that E. coli and probably many other prokaryotes require the tripeptide as protection against chemical challenges (e.g. reactive free radicals, oxidative stress, genotoxic agents and miscellaneous xenobiotics) . Similar conclusions can be drawn for microbial eukaryotes, although a minimum level of GSH might be required for growth, as previously shown for Sacch. cerevisiae. Detailed analyses of the effects of GSH deficiency on growth and respiration of yeast cells will probably lead to enlightenment of some of the regulatory functions of GSH in this organism.

E. PHYSIOLOGICAL ROLES OF y-GLUTAMYLTRANSPEPTIDASE

1. Transport of Amino Acids and Potassium Ions

In animal cells, y-GT is membrane bound and highly concentrated at sites of extensive amino-acid transport (Meister, 1983). The hypothesis that amino acids might be transported as y-glutamyl derivatives has been suggested by Meister and his coworkers. Recent findings indicate that formation and transport of y-glutamyl-amino acids across the cellular membrane is most likely associated with transport of amino acids by kidney cells, although the relative contribution of this pathway is still unknown (Meister, 1988).

The possible involvement of the y-glutamyl cycle in uptake of amino acids by Sacch. cerevisiae and Candida utilis has been proposed (Mooz, 1979; Osuji, 1980) but is further debated. A number of significant observations have been made regarding permeation system of amino acids and dipeptides into Sacch. cerevisiae (Wiame et al . , 1985). Although several systems that mediate amino-acid transport in yeast exist, the mechanism by which some operate is not yet known. In this connection, the idea that certain of these solutes might be transported into yeast cells as y-glutamyl

GLUTATHIONE IN MICRO ORGANlSMS 259

derivatives was appealing. To support this assessment, investigations have emphasized a requirement for studies of y-GT regulation, using selective inhibitors and different yeast mutants impaired in the amino-acid permea- tion system. As shown in Table 1, the intracellular level of y-GT remained unaffected in mutants which lost the general amino-acid permease (Gap-) and the specific systems for L-arginine (Argp-) and L-lysine (Lysp-) , but only very low activity was detected in the Apf mutant growing on urea, a nitrogen source promoting high levels of y-GT in the wild-type strain (Penninckx et al., 1980). The Apf mutation most likely affects a common element shared by all of the amino-acid permeation systems (Grenson and Hennaut, 1971). This latter result supported the idea that y-GT could be involved in a group-translocation mechanism similar to the HPr or protein I elements of the bacterial phosphotransferase system (Roseman, 1969; Kunding, 1976). However, the effect of the Apfmutation on the level of y-GT activity was further shown to be only indirect (Jaspers, 1985). No correlation can thus be established between the level of y-GT activity and mutations impairing bulk uptake of amino acids in yeast and no relation was found with turnover of GSH (Robins and Davies, 1981a,b). From the GSH content of yeast, its turnover and rate of L-glutamate uptake, it has been estimated that about 30-100 molecules of L-glutamate are transported for every degraded GSH molecule. Similar conclusions were reached by Jaspers and his colleagues (1985) using a different methodology for determination of GSH turnover. In addition, specific chemical inhibition of y-GT in whole Sacch. cerevisiae using 6-diazo-5-oxo-~-norleucine did not affect uptake rates of several amino acids, dipeptides and y-glutamyl compounds (Payne and Payne, 1984). Finally, progress in the field of amino- acid transport in micro-organisms, especially in Sacch. cerevisiae, has highlighted the role of ionic pumps (Eddy, 1982).

To conclude, all of these results militate against a role for the y-glutamyl cycle in transport of amino acids in yeast, but do not rule out, for instance, possible involvement of y-GT in a vacuolar-facilated transport (Jaspers and Penninckx, 1984) or in generation of y-glutamyl derivatives to act as signals in regulation of transmembrane processes (Payne and Payne, 1984). Discrete pools of y-glutamylglutamine and y-glutamylglutamate were indeed detected in Sacch. cerevisiae growing on glutamate as a nitrogen source (Jaspers et al., 1985). The rate of y-GT biosynthesis is derepressed in such a medium and L-glutamate, as well as L-glutamine, was shown to be a good acceptor in the transpeptidation reaction catalysed by the yeast enzyme (Penninckx and Jaspers, 1985). Substantial portions of intracellular amino acids and glutathione are compartmentalized within the vacuole. Redistribution of amino acids between compartments has been demon- strated in response to metabolic signals (Messenguy et al., 1980; Wiemken,

260 M J P F N N I N C K X AND M r F I S K ~ N S

1980; Kitamoto et al., 1988). To what extent y-glutamyl derivatives might play a role in such processes is unknown, and further research work along these lines is needed.

A role for GSH in transport of potassium ions in E. coli has been discovered (Meury and Robin, 1985, 1990). Mutants deficient in GSH, unlike the wild type, rapidly leak potassium ions and grow very slowly on a medium containing a low concentration of potassium ions. Addition of GSH, ophthalmic acid or y-(glutamylcysteiny1)bisglycine to the culture medium decreases the leak of potassium ions and restores a wild-type growth rate. A protein with a highly apparent affinity to GSH (Kd 50 n M )

was identified as a cytoplasmic element regulating the circulation of potassium ions. For each of three peptides, the Kd value €or binding is similar to the minimal concentration achieving maximal decrease of the leak. The possible physiologicai effectors might be y-glutamyl derivatives generated by periplasmic y-GT.

2. Mobilization of Glutathione as a Sulphur Source

Glutathione was long ago cited as a growth factor for micro-organisms but, meanwhile, few detailed reports have further addressed the subject (Brown, 1974; Penninckx and Jaspers, 1982). Glutathione also appeared to be an alternative source of sulphur for morghogenesis of spores of Bacillus cereus (Cheng et al., 1973) and vegetative cells of a species of cyanobacterium (Giddings et al., 1981). In Sacch. cerevisiae, substantial evidence indicates that the y-glutamyl cycle can function to meet an intracellular requirement for cysteine, and is plausibly involved in overall regulation of sulphur metabolism in this organism (Elskens et al., 1991). Potentially, two pathways exist for synthesis of cysteine (Jones and Fink, 1982; Ono et al., 1988). The first (Fig. 4, reactions (1)-(2)) proceeds by acetylation of serine to yield O-acetylserine (OAS), followed by a sulphydrylation. The latter enzyme also acts as O-acetylhomoserine (OAH) sulphydrylase (reaction (4); Yamagata, 1980). This pathway is analogous to the cysteine bio- synthetic pathway in enteric bacteria (Soda, 1987) and plants (Giovanelli, 1987) and has been referred to as direct sulphuration (Ono et al., 1991). In Sacch. cerevisiae, OAS-OAH sulphhydrylase was shown to be repressed by methionine (Cherest et al., 1969); the same has been shown for Neurospora crassa (Wiebers and Garner, 1967), Aspergillus nidulans (Paszewski and Grabski, 1974, 1975) and Saccharomycopsis lipolytica (Morzycka and Paszewski, 1982). The second pathway proceeds from homocysteine and involves the cystathionine biosynthesis cycle (reactions (5)-(8); Masselot and de Robichon-Szulmajster, 1975; Masselot and Surdin- Kerjan, 1977). This pathway is analogous to the mammalian cysteine

GLUTAT1 IIONE IN MICKO-ORGANISMS 26 1

methionine SAM

( 5 ) (3) (13) c ystathionine y O-acetylhomoserine + homoserine

t so 2-

(1) serine + 0-acetylserine P+

glycine + c ysteinylglycine

glutamate and"f-glutamyl- 4, (16)

cysteine glutamate

(14)

cysteine glutamate

(14)

"f -glutamylcysteine

peptides \ glutathione r /

FIG. 4. Pathways showing the main fluxes of sulphur-containing compounds in Saccharomyces cerevisiae (Elskens et ul., 1991). (1) indicates serine acetyltransferase, (2) cysteine synthase (OAS sulphydrylasc). ( 3 ) homoscrinc acctyltransferase, (4) homocystcine synthase (OAH sulphydrylase), ( 5 ) y-cystathionine synthase, (6) y- cystathionase (C7T y-lyase), (7) p-cystathionasc, (8) p-cystathioninc synthase, (9) homocysteine methyltransferase, (10) S-adenosylmethionine (SAM) synthetase, (1 1) S-adenosylmethionine dcmcthylasc, (12) adcnosylhomocystcinase, (13) a sulphatc-reducing pathway, (14) y-glutamylcysteine synthetase, (15) glutathione synthetase, (16) y-glutamyltranspcptidasc and (17) cysteinylglycinc dipeptidase.

biosynthetic pathway (Reed et ai., 1983; Griffith, 1987) and has been referred to as reverse transulphuration (Soda, 1987). Ono et al. (1991) have shown that the second enzyme, y-cystathionase (reaction (6); CTT y-lyase) is derepressed nearly 15-fold when Sacch. cerevisiae was starved for both inorganic and organic sulphur-containing compounds. Growing evidence supports, furthermore, that wild-type strains of Sacch. cerevisiae utilized methionine preferentially over sulphate if both were available in the environment. The enzyme CTT y-lyase is repressed in methionine- grown cells as well as in sulphate-grown cells, while derepression takes place following an extensive depletion of cysteine. From these observations, the authors inferred that CTT y-lyase is the target for regulation and that cysteine is very likely the effector in this regulation. Almost simultaneously, a working model for the main fluxes of sulphur-containing compounds in

262 M J PENNINCKX AND M T FI SKFNS

Sacch. cerevisiae involving GSH metabolism was proposed (Elskens et af . , 1991). It suggests that, in the presence of sulphate as a nutrient, most excess sulphur is incorporated into GSH (reactions (13)-(15)). Under these conditions, y-GT activity is low and the turnover rate of GSH is exceedingly slow. When cells are deprived of sulphate, GSH is apparently able to serve as an internal sulphur source until it reaches a residual concentration of about 10% of its normal value. An increase in the specific activity of y-GT is attained by derepressing synthesis of the enzyme and, as a consequence, the turnover rate of GSH increases (reactions (16)-(17)). Regulatory controls of y-GT and enzymes of cysteine biosynthesis, i.e. CTT y-lyase (reverse transulphuration) and OAS-OAH sulphydrylase (direct sulphura- tion), appeared distinct and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed. As already described, derepression of y-GT correlated best with a decrease in the GSH pool among examined sulphur compounds, which was not so for C7T y-lyase and OAS-OAH sulphydrylase. The model requires, moreover, that derepression of the bidirection transulphuration pathway leads to a more active transfer of sulphur atoms from cysteine to methionine and vice versa (Elskens et al., 1991). Hence, it could be argued that Sacch. cerevisiae is able (a) to avoid excess biosynthesis of cysteine by direct sulphuration by regulation of OAS-OAH sulphydrylase (Cherest et al., 1969), (b) to prevent back-flow from methionine through reverse transulphuration by regulation of CTT y-lyase (On0 et af., 1991), and (c) to utilize GSH as a physiological reservoir (source and sink) for cysteine. Yeast y-GT is a vacuolar enzyme and, since about 50% of the total cellular GSH is concentrated in the central vacuole (Jaspers and Penninckx, 1984), it has been suggested that the tripeptide might have a storage function, similar to that for glycogen, trehalose, polyphosphates and lipids (Gancedo and Serrano, 1989), and be mobilized during starvation andlor reproduction (Elskens et a f . , 1991).

IV. Interconversion of Glutathione and Glutathione Disulphide

Intracellular GSH is converted to GSSG by selenium-containing GSH peroxidase, which catalyses destruction of hydrogen peroxide and organic peroxides (see Section 1V.B). A selenium-independent form, active with numerous organic hydroperoxides, was also found and is related to certain GSH S-transferases, a group of cytosolic isoenzymes with overlapping substrate specificity (see Section V.A). Glutathione transhydrogenases (protein disulphide isomerase, thiol-transferase, thiol-disulphide oxido- reductase), a group of proteins involved in thiol-disulphide exchanges,

GLUTATHIONE IN MICRO-ORGANISMS 263

catalyse also formation of GSSG. A number of reactions of this type will be further considered (see Section 1V.A). There is substantial evidence that chemical oxidation of GSH occurs within the cell by non-enzymic processes. Amongst the most useful agents to oxidize intracellular GSH are diazene derivatives introduced by the Kosowers (1978a). Formation of GSSG by reactions with free radicals, and oxidation of GSH mediated by trace metals, have been reported as well, although the exact mechanism still remains obscure (see Section 1V.C). Finally, reduction of GSSG is catalysed in vivo by the widely distributed GSH reductase, whose role in the GSH redox cycle is particularly important (see Section 1V.C).

A . GLUTATHIONE TRANSHYDKOGENASES

By virtue of its thiol group and its relatively high concentration in cells, GSH has long been thought to play a leading role in the thiol status of many cell constituents, including proteins with cysteine residues (Baron, 1951; Jocelyn, 1972). Several proteins depend upon disulphide bonds for stability of their folded conformation, while many metabolic and physio- logical functions involve thiol-disulphide exchanges, e.g. protein synthesis and degradation, activation and inactivation of enzymes, synthesis of deoxyribose intermediates, mitosis, and alterations in the dormant state of bacteria (Flohe and Gunzler, 1976; Fahey, 1977; Kosower and Kosower, 1978; Freedman, 1979; Buchanan, 1980; Mannervik and Axelsson, 1980).

Regulation of enzymes by thiol-disulphide exchanges was recognized a long time ago (Baron, 1951). The simplicity of the mechanism for covalent modification of enzymes is especially appealing since the reaction is readily reversible and quite well characterized (Szajewski and Whitesides, 1980; Creighton, 1983). Formation of the mixed disulphide of an enzyme can either increase or decrease catalytic activity, and regulation of several enzymes by this mechanism has been previously reported (see Ziegler et al. , 1983). However, since the initial model studies of these processes in vitro were much slower than expected from in vivo kinetics of synthesis, folding and secretion of disulphide-bonded extracellular proteins (Anfinsen, 1973), there was little doubt that thiol-disulphide exchanges were most likely catalysed in vivo by thiol transferase or transhydrogenase enzyme(s). In the course of research for a potential catalyst for disulphide formation in proteins, a disulphide isomerase activity (PDI: EC 5.3.4.1) was discovered in studies on reactivation of reduced pancreatic RNAase (Goldberger et al., 1963). Concurrently, with Anfinsen’s group, Tomizawa (1962) purified an enzyme from bovine liver that catalyses reductive cleavage of disulphide bridges in insulin in the presence of GSH. The essential similarity of these two thiol-protein disulphide exchange reactions was noted by several

264 M J PENNINCKX AND M T ELSKENS

investigators, and the question of identity was raised when Ansorge and his coworkers demonstrated that both activities copurified in their rat-liver preparations (Ansorge et al., 1973a,b). Nevertheless, because so many varied substrates participate in thiol-disulphide exchange reactions, there may be several thiol transferases or transhydrogenases. In an attempt to identify the enzyme(s) responsible for thiol-protein exchange, Freedman (1979) considered the idea that there may be separate enzymes with different but overlapping specificities. Although a great deal of work has been carried out since then on details of folding and oxidation of reduced proteins in vitro, while special attention has been directed towards systems catalysing such reduction and rearrangement of protein disulphide bonds in vivo (see Freedman et a f . , 1988), the physiological functions associated with these proteins are not fully understood.

In prokaryotes, Sundquist and Fahey (1989) demonstrated that GSH thiol transferase was produced by Escherichia coli and Beneckea alginolytica in substantial amounts (higher than in rat liver) but was absent from other investigated GSH-producing bacteria such as Rhodospirillum rubrum, Chromatium vinosum and Anabaena sp. In halobacteria, which produce the dipeptide y-glutamylcysteine instead of GSH, the presence of significant thiol transferase activity was also reported. Based upon these results, the authors have suggested that GSH and the thiol transferase may play a significant, although not universal, role within cells in alleviating disulphide formation. Interestingly, numerous heterotrophic bacteria isolated from soil, water, sediment and vegetation, and marine algae were found to methylate sulphide. Production of methanethiol was shown to depend on a thiol methyltransferase activity that uses organic thiols as substrates. The enzyme is widespread in bacteria and may contribute to biogenic emission of methylated sulphur gases and production of methyl thioethers (Drotar et al., 1987). The specificity of the protein(s) towards glutathione is, however, unknown. Other possible functions of significant ecological importance for bacterial protein-disulphide transhydrogenase have been recently proposed (Pel and Gottschal, 1989). Fermentation of chitin by mixed cultures of a chitinolytic Clostridium sp. and various non-chitinolytic bacteria was shown to proceed up to eight times faster than in pure cultures of the Clostridium strain. The agent responsible for this stimulation was found either in spent media of the mixed culture or in cell-free extracts of the non-chitinolytic bacteria and was thought to be a thioredoxin or a thiol- disulphide transhydrogenase. This stimulation thus emphasized a possible new type of interspecies interaction in anaerobic cultures.

A thiol-disulphide transhydrogenase with a relatively high affinity and reaction rate to cystine was purified from yeast several years ago (Nagai and Black, 1968). The enzyme was inactive towards insulin and other

Glutamate + cysteine

I y GLUTAMYL C Y S ” E SYNlHETASE (Gslul )

y-Glutamylcysteine

GLUTATHIONE S Y I V l W T m (GshB )

GLUTAREDOXIN (Grx 1

7 Glycine

GLUTATHIONE m u c r ~ ~ ( G ~ K 1- -

Ribonucleotide reduction Sulphate reduction Disulphide reduction > Metliionine sulphoxide reduction

NADPH

THIoREDoxlN L THIoREDoxlN REDuCrksE(Tr*B) ( T d )

FIG. 5. Pathway showing transfer of electrons in Escherichia coli by the thioredoxin and the glutaredoxin systems. From Fuchs et ul. (1983).

266 M. J . PENNINCKX A N D M T. ELSKENS

proteins containing disulphide bridges. Quite recently, the nucleotide sequence of the yeast YCL313 gene, localized on the left arm of chromo- some 111, was determined (Scherens et al., 1991). This gene encodes for a protein with 522 amino-acid residues (MT 58,300) which presents large homologies with the human, mouse, chicken, bovine and rat PDI proteins. Furthermore, all of these proteins contain two regions, defined as a and a', with strong similarity to the thioredoxin active sites. It was thus suggested that the YCL313 gene encodes for a yeast PDI protein. Gene disruption of YCL313 leads to a lethal phenotype, indicating that this gene is essential for cell survival.

Although GSH transhydrogenases probably have important functions related to synthesis, structure, degradation and folding of proteins, other systems that affect the thiol-disulphide status of cells are also of considerable importance. These include the widely distributed thioredoxin and glutaredoxin systems (Holmgren, 1983, 1985; Holmgren et al., 1986; Gleason and Holmgren, 1988).

R . THE GLUTAREDOXIN SYSTEM

Figure 5 illustrates transfer of electrons by thioredoxin and glutaredoxin systems in micro-organisms. Ribonucleotide reductase catalyses the first step in DNA synthesis by reducing four different ribonucleotides to the corresponding deoxyribonucleotides (Reichard and Thelander, 1979; Reichard, 1987). Reduction of the ribose moiety of a ribonucleotide requires a hydrogen-donor system, and thioredoxin from E. coli was the first possible physiological hydrogen donor for the ribonucleotide reductase described by Reichard and his coworkers (Mathews et al., 1987). Thioredoxin is a small ( M , 12,000) ubiquitous redox protein with the conserved active-site structure of -Trp-Cys-Gly-Pro-Cys- (Gleason and Holmgren, 1988). The oxidized form (TRX-S2) contains a disulphide bridge which is reduced to the dithiol form by NADPH and the FAD enzyme thioredoxin reductase. The reduced form (TRX-(SH),) is a powerful protein-disulphide oxidoreductase. Thioredoxin has been characterized from a wide variety of prokaryotes, microbial eukaryotes, plant, and animal tissues and appears universal (Holmgren, 1981; Holmgren et al., 1986; Gleason and Holmgren, 1988; Meyer et al., 1991). Further discussion on the biochemical aspects of this protein is outside the scope of this review.

During studies of thioredoxin mutants in E. coli that were killed by phage T7 infection (Mark and Richardson, 1976), glutaredoxin, another hydrogen donor system for ribonucleotide reduction requiring GSH, was discovered (Holmgren, 1976). A remarkable account of the relative contribution, structural relation and distribution of these two hydrogen-donor systems

GLUTATHIONE IN MICRO-ORGANISMS 267

in E. coli was made by Holmgren and his coworkers. Isolation and characterization of E. coli mutants which are impaired either in the thioredoxin (Fuchs, 1977; Mark et al., 1977; Fuchs et a f . , 1983) or glutaredoxin (Kren et al., 1988; Russel and Holmgren, 1988; Sandberg et al., 1991) system have, furthermore, extended the field of investigation from previous in vitro to in vivo studies.

Glutaredoxin from E. coli is a heat-stable acidic protein with about 85 amino-acid residues with a cystine disulphide bridge and a molecular weight of 9674 for the reduced form, making it one of the smallest known enzymes (Hoeoeg et al., 1983). The single active-centre disulphide has the structure -Gly-Cys-Pro-Tyr-Cys-, with the half-cystine residues located at positions 11 and 14 in the polypeptide chain (Holmgren, 1983). Reduction of glutaredoxin is readily obtained with dithiothreitol and it is thereby similar to thioredoxin (Holmgren, 1979). However, neither protein shows immuno- logical cross-reactivity , and both are structurally unrelated and are apparently different gene products (Holmgren, 1976, 1983; Hoeoeg et al., 1986). Unlike thioredoxin, which requires a dithiol (dihydrolipoic acid or dithiothreitol) as a reductant, reduction of the disulphide bridge in glutaredoxin is obtained by GSH in the presence of NADPH and glut at hione reductase :

CDP + 2GSH- dCDP + GSSG + H20

GSSG + NADPH + H+-+ 2GSH + NADP'

(glutaredoxin)

(glutathione reductase)

Chemically reduced glutaredoxin is enzymically active in conversion of each of the four ribonucleoside 5'-phosphates in the presence of ribonucleotide reductase (Holmgren, 1979). Oxidized glutaredoxin is not a substrate for NADPH and thioredoxin reductase, and thioredoxin does not catalyse GSH reduction, demonstrating that thioredoxin and glutaredoxin systems operate independently (Fig. 5). However, since the structure of glutaredoxin was shown to be quite similar to phage T4 thioredoxin, with respect to the size of the polypetide chain and its amino acid-residue sequence, it was suggested that the T4 protein-encoding gene might have evolved from an early glutaredoxin gene (Hoeoeg et al., 1983). The mixed properties and functional similarities of these two proteins are compatible with such a view. Given present knowledge of the structure of thioredoxin and glutaredoxin, an attempt was made to deduce some common features at both the primary and tertiary structural levels that might reflect common functional properties. There are great differences between the sequences of thioredoxins from E. coli and bacteriophage T4, but the tertiary structures of both proteins are quite similar in spite of their poor sequence homology (Branden et al., 1983). A comparison of the

268 M. I. PENNINCKX A N D M. '1. ELSKENS

tertiary structure gave a common fold of 68 a-carbon atoms with a mean square root difference of 2.6 A (Eklund et al., 1984). Assuming that glutaredoxin has the same common fold, the amino acid-residue sequence of glutaredoxin has been aligned to those of thioredoxins from E. coli and phage T4. A model of the glutaredoxin molecule was built on a vector display using this alignment and the phage T4 tertiary structure (Eklund et al., 1984). By comparison of this model with those of thioredoxins, the authors have identified a common molecular surface area on one side of the redox active disulphide bridge which might represent the binding region for redox interactions with other proteins (e.g. ribonucleotide reductase). In spite of the fact that there are some arguments for the existence of a glutathione-binding site on ribonucleotide reductase (Hoeoeg et al., 1982), the functional organization with glutaredoxin and (or) thioredoxin in cells remains largely unknown.

While deoxyribonucleotides are apparently formed by ribonucleotide reductase, the nature of the in vivo hydrogen donor is still questionable (Holmgren, 1988). It should be stressed that, if the apparent concentration of thioredoxin in wild-type E. coli is about SO-fold higher than that of glutaredoxin, the molecular activity of the glutaredoxin system seems greater (Holmgren, 1979, 1983). Quite possibly, both thioredoxin and glutaredoxin might function during normal growth and serve as substitutes for each other. Studies with E. coli mutants impaired in the thioredoxin or glutaredoxin system support this view. A strain deficient in thioredoxin reductase (TrxB) , which was unable to use methionine sulphoxide as a methionine source, was isolated by Fuchs (1977). In permeabilized whole cells of the mutant, the ribonucleotide reductase assay revealed only 5% of the parental activity. However, when the cell preparation was supplemented with GSH, reduction of uridine diphosphate was observed at the same rate as that of the parental strain, indicating that glutaredoxin could replace thioredoxin reductase. The TrxB mutant grew like the wild type under laboratory conditions, and the mutation mapped between 14 and 16 minutes on the chromosome of E. coli (Fuchs et al., 1983). A thioredoxin (TrxA)-defective mutant showing no altered growth characte- ristics was also isolated (Mark et al., 1977). The mutation mapped at 84 minutes on the Chromosome and was 34"% contransducible with the MetE gene. However, transduction experiments using this TrxA strain have indicated the existence of several genetic defects linked with the mutation. Due to close linkage of deleterious mutations, and possibly a structural alteration in the region containing the TrxA gene, it has proved difficult so far to interpret the phenotypes of thioredoxin and GSH-deficient double mutants.

A glutaredoxin-negative mutant of E. coli was recently constructed by

G I IJ I A l H I O N F IN MI( KO ORGANISMS 269

inactivation and insertion of a 2 kb kanamycin-resistance fragment into the coding sequence of the glutaredoxin gene in E. coli (Hoeoeg et al . , 1986). The inactivated gene was inserted into the chromsome of E. coli and mapped at about 18 minutes. A gene-replacement technique was then used to obtain a mutant that lacked glutaredoxin, as tested by radio-immunossay and ribonucleotide reductase assay (Russel and Holmgren, 1988). While obtaining Grx- or Trx- mutants showed that glutaredoxin or thioredoxin, taken alone is not essential for viability of E. coli, attempts to construct a double mutant lacking both glutaredoxin and thioredoxin have been unsuccessful. This indicates, at least, that the presence of one of the two electron donors is essential for growth (Russel and Holmgren, 1988). Glutaredoxin-defective mutants of E. coli were also characterized by Fuchs and his coworkers (Kren et al., 1988). The mutants have a detectable but decreased glutaredoxin-ribonucleotide reductase activity in either crushed or permeabilized cells. The mutants appeared deficient in sulphste and ribonucleotide reduction, suggesting that in vivo glutaredoxin is the preferred cofactor for ribonucleotide and adenosine-3’-phosphate-5’- phosphosulphate reductases.

C. GLUTATHIONE PEROXIUASE AND THE ANTIOXIDANT DEFENCE SYSTEM

I N MICRO-ORGANISMS

Hydrogen peroxide is a ubiquitous biological compound, formed as the enzymic product of numerous oxidases (e.g. superoxide dismutase) present in the cell cytosol, plasma membranes, peroxisomes and mitochondria1 matrix, as well as by auto-oxidative reactions of haemoproteins, ilavo- proteins and other cell components (Chance et al., 1979; Fridovitch, 1982; Kappus, 1986). Disposal of hydrogen peroxide is of primary importance with regard to oxidative injuries, since it is a component of both the Fenton and Haber-Weiss cycle reactions (Kappus, 1985; Halliwell and Gutteridge, 1990), which are possible sources of the reactive hydroxyl radical. This radical is one of the most reactive oxygen metabolites and is thought to be responsible for serious damage that can occur during redox cycling processes, e.g. peroxidation of membrane lipids, protein and DNA. Lipid peroxidation disrupts membrane functions and yields toxic reactive by- products, such as malonic dialdehyde or 4-hydroxynonenal (Kappus, 1987; Halliwell, 1991). Protein damage leads to amino-acid oxidation, resulting in structural changes and enzyme inactivation (Dean et al., 1986). Damage to DNA leads to strand breakage, deoxykbose fragmentation and extensive chemical alteration of purine and pyrimidine bases (Von Sonntag, 1987; Halliwell and Aruoma, 1991). Thus, intracellular generation and metabolism of hydrogen peroxide has to be limited by the presence of very

270 M J PENNINCKX AND M T ELSKFNS

efficient systems in cells for its decomposition. Cellular destruction of hydrogen peroxide was found to be catalysed by different enzymic systems, among which the most studied are catalase (hydrogen-peroxide oxidoreductase (EC 1.11.1.6) and GSH peroxidase (EC 1.11.1.9); Kappus, 1986; Halliwell and Gutteridge, 1989). A study of inborn errors of metabolism strongly suggests, furthermore, that GSH peroxidase is most important in removing hydrogen peroxide from human tissues, probably because it is located in the same subcellular compartments as superoxide dismutase (Halliwell, 1991).

The chemical and physical properties of GSH peroxidase have been reviewed by Wendel (1980). The enzyme fails to display saturation kinetics with respect to GSH concentration, and the extrapolated V,,, value is consequently infinite. Lack of a defined K, value agrees with the fact that the apparent maximum velocity for infinite peroxide concentration is a linear function of GSH concentration, thereby supporting a ter uni ping- pong mechanism (Wendel, 1980).

In biological systems, glutathione peroxidase activity is expressed by at least two enzymes (Lawrence and Burk, 1976). These are the selenium- dependent form containing a selenocysteine residue at the active site and which is able to reduce hydrogen peroxide and organic hydroperoxides (Flohe et al., 1980), and the selenium-independent form which acts mainly on organic hydroperoxides and is related to certain GSH S-transferase izoenzymes (Lawrence and Burk, 1978; Prohaska, 1980). In addition, GSH peroxidation reactions can occur by non-enzymic processes. Several low molecular-weight compounds (e.g. organoselenium compounds and dithio- carbamates) were found to have a GSH peroxidase-like activity. Confusion may occur if unfounded generalizations are made, and a number of reactions of this type will be further discussed. According to Ziegler and his colleagues (1983) , peroxidation of glutathione is capable of altering the intracellular thiol-disulphide balance, and is undoubtedly a major source of cellular disulphide, although most of the GSSG formed is reduced in turn by GSH reductase and NADPH. Consequently, under oxidative challenge, these bioreduction reactions are able to consume a significant fraction of NADPH reducing equivalents in the cell. As described later, GSH can be assigned a regulatory role in these processes due to the high demand that can be placed on cellular capacity to generate NADPH (Reed, 1986).

While GSH peroxidase is widely distributed in animal tissues, its occurrence in micro-organisms is still questionable. Representative species of the most important GSH-producing bacteria, e .g. the purple bacteria and cyanobacteria, (B . alginolytica, R. rubrum, C. vinosum and Anaebaena sp. strain 7119) and E. coli were found to lack any significant GSH peroxidase and transferase activities. Similarly, GSH peroxidase could not

GI U IAI'lIIONE IN MICRO-ORGANISMS 27 1

be detected in halobacteria which produce the dipeptide y-glutamylcysteine (Sundquist and Fahey, 1989). The virtual absence of GSH peroxidase from photosynthetic bacteria raises, of course, serious questions concerning the antioxidative potential of cells to scavenge hydroperoxides formed as by-products of photosynthetic activity. It has been suggested that in cyanobacteria (Nostoc muschorum and Synechococcus spp.) the effective mechanism for removal of hydroperoxides involves ascorbate peroxidase and recycling of GSH and ascorbate (Tel-Or et al., 1985). That GSH might sustain production of the reducing equivalents in these processes through action of a GSH reductase-NADPH-catalysed reaction was also emphasized by Dupouy et al. (1985).

The presence of a GSH peroxidase activity was demonstrated in strains of Pseudomonas putida, especially in cells oxidizing trivalent arsenite. Arsenite is thought to initiate free-radical lipid peroxidation yielding malonic dialdehyde. Accumulation of malonic dialdehyde was shown in experiments with cell homogenates, while the activity of antioxidant enzymes (superoxide dismutase, catalase, GSH peroxidase and reductase) rose in bacteria grown on an arsenite-containing medium. It was, therefore, suggested that these enzymes may interfere with the free-radical processes allowing metabolization of arsenite (Abdrashitova et al., 1990).

Unlike bacteria, GSH peroxidase appears to be much more widely distributed in micro-algae (Overbaugh and Fall, 1982). Euglena gracilis was shown to possess both types of GSH peroxidase activity. The enzymes were apparently not induced in response to stimulation of cellular processes that generate oxidant species, such as p-oxidation or photosynthesis, but the peroxidation activity increased in autotrophic cultures containing the herbicide N'-(3,4-dichlorophenyl)-N,N-dimethylurea (Overbaugh, 1985). A selenium-independent peroxide was purified to electrophoretic homo- geneity from a permanently bleached strain of E. gracilis. The native enzyme has a molecular weight of 130,000, as measured by gel-permeation chromatography, and consists of four identical subunits ( M , 31,500), as indicated by SDS-PAGE (Overbaugh and Fall, 1985). A selenium- dependent form, whose enzymic properties were closely similar to the GSH selenium peroxidase found in animal tissues, was demonstrated in the green alga Chlamydomonas reinhardtii. (Yokota et al., 1988). Growth of the alga in a sodium selenite-containing medium increases the level of GSH peroxidase at the expense of ascorbate peroxidase. In contrast, two unicellular marine algae (Dunaliella primolecta and Porphyridium cruentum) have been found to contain a selenium-inducible, but non- enzymic, GSH peroxidase activity when cultured in the presence of selenite (Gennity et al., 1985a,b). Since part of the hydrogen peroxide and t-butyl- hydroperoxide (t-BuO0H)-dependent oxidation of glutathione in cell

272 M J PFNNINCKX AND M T ELSKENS

homogenates was shown to be heat- and cold-stable, it was suggested that this contribution could be non-enzymic in nature and probably mediated by a variety of small molecules such as ascorbate, selenocystine, selenomethionine, dimethylselenide and copper nitrate (Gennity et al. , 1985a). Crushed cells of the red alga Po. cruentum also contained, however, a heat- and cold-labile t-BuOOH-dependent reductase activity which accounted for about 90% of the total peroxidation activity. This possible enzymic activity remained unaltered by growth in the presence of selenite (Gennity et al., 1985a). To what extent both non-enzymic and enzymic processes might contribute to in vivo GSH peroxidation is still unknown, and there is no current evidence for an in vivo functioning of selenium as an antioxidant in these algae (Gennity et al., 1985b).

The antioxidant defence system of protozoa has also been investigated (Murray et al., 1981; Penketh and Klein, 1986; Fairfield et al., 1988; Fairlamb, 1990). In the human malarial parasite Plasrrzodium falciparum, three oxidant defence enzymes whose activity changed with the growth stages have been characterized (Fairfield et al., 1988). Isolated early intra- erythrocytic stages of P1. falciparum were shown to contain mainly catalase, superoxide dismutase and little, if any, GSH peroxidase activities, while late intra-erythrocytic stages were shown to possess much more of the dismutase and GSH peroxidase, and slightly less catalase. It was suggested, furthermore, that P1. faciparum and PI. berghei probably acquired most of their dismutase from the host, since the parasite-associated enzyme was predominantly cyanide-sensitive like the host enzyme, while parasites grown in red cells that had been partially depleted of superoxide dismutase were most sensitive to exogenous superoxide. Most trypanosomatids apparently lack GSH peroxidase (Penketh and Klein, 1986; Penketh et al., 1987). Instead, an analogous trypanothione peroxidase activity has been identified in Trypanosoma brucei and in Crithidia fasciculata (Henderson et al., 1987). It is thus possible that, in these organisms, the trypanothione reductase-peroxidase system has assumed the role of the GSH reductase- peroxidase system of mammalian cells. The situation is, however, less clear in Trypanosoma cruzi, where unstable ascorbate and low alkyl peroxidase activities have been detected, the latter probably being a GSH S-transferase (Yawetz and Agosin, 1981).

Glutathione peroxidase was found in cell-free extracts of Mucor spp., where enzyme production was shown to be almost completely associated with mycelial growth (Aisaka et al., 1983), and in Pyricularia oryzae, where the antioxidative systems (superoxide dismutase, GSH peroxidase and catalase) were thought to be involved in parasite tolerance (Nikolaev et al . , 1989). Controversial results were, however, reported regarding the presence of GSH peroxidase in yeasts. Aisaka and his colleagues (1983)

GLUTATHIONE IN MICRO-ORGANISMS 273

performed a systematic screening for the occurrence of GSH peroxidase in numerous micro-organisms and did not find any significant activities of this enzyme in any one of 31 yeast strains. In Saccharomyces cerevisiae, the enzyme was not detected by Smith and Shrift (1978) or Penninckx and Jaspers (1982), but was reported by Galiazzo and his colleagues (1987). Other investigations have emphasized the presence of GSH peroxidase activities involving both selenium-dependent and -independent forms in different yeast species, such as members of the genera Candida, Saccharomyces, Schizosaccharomyces, Hansenula and Sporobolomyces (Casalone et al., 1988). Nevertheless, the physiological contribution of GSH peroxidase to the defence systems of yeasts against peroxidative attacks needs to be clarified. Owing to its ability to grow either anaerobically or aerobically, Sacch. cerevisiae has been considered long ago as a particularly suitable biological model for investigating the cytotoxic effects of oxygen. A role for GSH peroxidase in these processes was apparently not previously envisaged (Sels and Brygier, 1980; Van Huffel and Sels, 1987). To identify some of the physiological parameters that can modulate cellular defences in Sacch. cerevisiae, Sels and his coworkers used different redox compounds known to potentiate in situ oxygen toxicity. From these results, it was obvious that both catalase and superoxide dismutase appeared essential for survival of the yeast under aerobic conditions. Quite recent studies have, however, demonstrated that, besides catalase, other peroxidase(s) might play a significant role in the removal of hydrogen peroxide (Verduyn et al., 1988). A catalase-negative mutant of Hansenula polymorpha was found to dismutate hydrogen peroxide generated intracellularly during oxidation of methanol and to destroy exogenous peroxides added to the culture medium. Destruction of hydrogen peroxide was apparently not attributable to any GSH peroxidase activity, but rather to a cytochrome- c peroxidase (EC 1.11.1.5) that increased to very high levels in cells growing on a glucose-hydrogen peroxide medium. A similar trend was observed with the wild-type strain of H . polymorpha, where an increase in the level of both cytochrome-c peroxidase and catalase activities was shown. By comparison, when Sacch. cerevisiae was grown in the glucose-hydrogen peroxide medium, the activity of catalase remained low because of the repressive effect mediated by glucose in this strain, while the cytochrome- c peroxidase activity rose with increasing rates of hydrogen peroxide utilization. Therefore, according to Verdyun et al. (1988), the peroxidase might be a key enzyme in detoxification of hydrogen peroxide in yeast, and catalase and the peroxidase might effectively compete for peroxidation reactions. However, cross-reactivity would probably be limited because of subcellular compartmentalization of the enzymes, as catalase is mainly located in peroxisomes whereas cytochrome-c peroxidase is mainly

27 4 M. 1. PFNNINCUX AND M. T. ELSKENS

mitochondrial. Although it appears that GSH peroxidase is apparently not implicated in the above-mentioned scavenging mechanisms, its possible involvement in a cellular defence system against lipid peroxidation cannot be ruled out. So far, except with some eukaryotic algae (Overbaugh, 1985), only very few studies have been devoted to the role of GSH peroxidase in microbial antioxidant defence systems.

D. THE GLUTATHIONE REDOX CYCLE

I . Glutathione Reductase

Glutathione reductase (EC 1.6.4.2; GSSG + NADPH + Hf+ 2GSH + NADP') is one of an important group of flavoenzymes about which a considerable amount of mechanistic and structural information is avail- able (Ghisla and Massey, 1989). This group of enzymes includes dihydrolipoamide dehydrogenase, GSH reductase, thioredoxin reductase, trypanothione reductase and mercuric reductase. Early work on the first three of these enzymes has been described in detail in a review by Williams (1976). With the exception of thioredoxin reductase, which appears to have a quite different protein structure, there are remarkable similarities between the known enzymes of the group (Ghisla and Massey, 1989). In the first place, a two-electron reduction of the enzymes yields a spectrally characteristic red intermediate which is a charge-transfer complex between a thiolate anion of one of the nascent cysteine residues and the oxidized flavin. Secondly, these enzymes have similar amino acid-residue sequences and chain folding, resulting in overall comparable three-dimensional structures. In spite of the fact that this considerable homology suggests that these proteins have evolved from a common gene ancestor, they currently fulfil distinct physiological functions and present different substrate specificities with little, if any, cross-reactivity (Williams, 1976).

Glutathione reductase has been characterized in a wide variety of micro- organisms and appears to be of universal occurrence (Williams, 1976; Ondarza et al., 1983; Serrano et al., 1984; Scrutton et al . , 1987; Montero et al., 1988; Sundquist and Fahey, 1989). However, two different GSH reductases have been isolated from bacteria: a NADH-specific enzyme from C. vinosum (Chung and Hurlbert, 1975) and a NADPH-specific enzyme from E. coli (Williams and Arscott, 1971). A reductase that is specific for NADH, the disulphides of pantetheine 4' ,4"-diphosphate and CoA has also been detected in many Gram-positive eubacteria and has been purified from Bacillus megaterium (Swerdlow and Setlow, 1983). Although these enzymes demonstrate different substrate specificities, they catalyse analogous reactions and share several physical characteristics. It

GI UTATHIONE IN MICRO-ORGANISMS 275

has been suggested, therefore, that GSH reductase might have evolved in bacteria from lipoamide dehydrogenase, perhaps subsequent to the appearance of an oxidizing atmosphere (Fahey, 1977; Fahey et al., 1987). Lipoamide dehydrogenase has, indeed, a much wider distribution among bacteria than any other disulphide reductase (Danson et al., 1984) and displays a considerable sequence homology with GSH reductase (Williams, 1976). It follows that substrate specificities of GSH reductase might type different stages in evolution of GSH metabolism, as C. vinosum appeared to be the least oxygen-adapted organism able to produce GSH in substantial quantities (Fahey et al., 1987). On the contrary, GSH reductase purified from photosynthetic cyanobacteria (Serrano et al., 1984) and, in a more general way, from eukaryotes (Williams, 1976), exhibits a considerable preference for NADPH over NADH and is quite specific towards GSSG. For example, the preference of the enzyme from Sacch. cerevisiae for the NADPH-GSSG reductase reaction was shown to be kinetically related to the high catalytic efficiency and low dissociation constants of the substrates (Tsai and Godin, 1987). Cloning of the gor genes encoding GSH reductase in E. coli (Greer and Perham, 1986) and Pseudomonas aeruginosa (Perry et al., 1991), and site-directed mutagenesis, have further shown the similarities (and differences) between GSH reductases from several sources and also allowed determination of the amino acid-residue sequence involved in substrate specificity. The three-dimensional structure of the enzyme from E. coli, which was solved at the 3 A level, displayed a considerable homology with the well-investigated human enzyme (Pai and Schulz, 1983; Karplus and Schulz, 1987) and also showed some spectacular effects of site-directed mutations, such as a change in the cofactor specificity from NADPH to NADH (Ermler and Schulz, 1991).

Among other related proteins most likely having a similar physiological function to GSH reductase are a bis-y-glutamylcysteine reductase, purified from Halobacterium salinarum (Sundquist and Fahey, 1988) and trypanothione reductase (Shames et al., 1986; Henderson et al., 1987; Krauth-Siege1 et al., 1987). The gene coding for the latter enzyme has also been isolated and cloned from the cattle pathogen Trypanosoma congolense and Leishmania donovani (Shames et al., 1988; Taylor et al., 1989). In both studies the two disuphide-specific reductases were thought to play an important role in maintaining cellular thiol groups in a reduced state in organisms that are either lacking GSH, but producing the dipeptide y- glutamylcysteine such as H. salinarum, or exhibiting a peculiar and unique metabolic trypanothione pathway (see Section 11).

Steps involved in the reaction catalysed by GSH reductase have been dissected in detail for the enzyme from baker’s yeast by a combination of classical spectroscopic examination, fast kinetics, isotope effects and by

276 M J PFNNINCKX AND M T FISKFNS

specific chemical modifications (Ghisla and Massey, 1989). A hybrid kinetic mechanism (bi-bi ordered sequential and ping-pong) has been proposed for the enzymes from yeast (Mannervik, 1973), E. coli (Perham et al., 1988), Anabaena sp. (Serrano et al., 1984) and Phycomyces blakesleeanus (Montero et al., 1990). It is particularly impressive that the balance flux between the ping-pong and ordered sequential pathways can be infl7ienced by a mutation of just one amino-acid residue, probably a tyrosine residue, as shown with the protein from E. coli (Perham et al., 1987). As with other reductases, such as nitrate, nitrite and NADP' reductases, GSH reductase is inactivated after reduction by its own electron donor, NADPH, and other reductants. Inactivation of the purified enzymes from Sacch. cerevisiae (Pinto et al., 1984, 1985) and E. coli (Mata et al., 1985a) was shown to be a time-temperature and pH-dependent process. Since both active and inactive forms of the enzymes had similar molecular weights, inactivation was attributed to intramolecular modification(s). The purified enzyme from Sacch. cerevisiae displayed protection against redox inactivation in the presence of GSSG, ferricyanide, GSH and dithiothreitol. High concentrations of NADP+ and GSSG effectively protected the enzyme at even lower concentrations than that required by GSH. It has been suggested that this auto-inactivation of GSH reductase by NADPH and its subsequent reactivation by GSSG has an important in vivo regulatory role (Lopez- Barea and Lee, 1979; Pinto et al., 1983). Redox interconversion of the enzyme was further demonstrated using crushed and permeabilized E. coli, treated with different reductants, and with intact cells incubated with compounds known to alter the intracellular redox state. In both sets of experiments, the results indicated that the interconversion mechanism was most likely controlled by intracellular NADPH and GSSG concentrations (Mata et al., 1985b).

The level of GSH reductase activity may thus reflect the physiological need of the cells and could, presumably, regulate its own requirement for NADPH. It is known that reducing equivalents needed in the NADPH- dependent reactions of anabolism are derived from substrates of several dehydrogenases. In yeasts, Bruinenberg et al. (1983a) have shown that the NADPH requirement for biomass formation is strongly dependent on available sources of carbon and nitrogen and it was inferred that the carbon flow towards NADPH-producing pathways should vary accordingly. Most likely, the hexose monophosphate pathway, and possibly NADP+-linked isocitrate dehydrogenase, would be the major sources of NADPH in Candida utilis (Bruinenberg et al., 1983b). Glucose-6-phosphate dehydrogenase, which catalyses the first reaction on the hexose mono- phosphate pathway, is inhibited by NADPH (Bonsignore and De Flora, 1972; Llobell et al., 1988). Regulation of the enzyme was expected since

GLUTATHIONE IN MICRO-ORGANISMS 277

the ratio of NADPH to NADP' was found to vary with respect to the physiological status of the cell. Efforts to understand regulation of the hexose monophosphate pathway led Eggleston and Krebs (1974) to consider the ability of GSSG to counteract inhibition of NADPH on glucose-6- phosphate dehydrogenase, and this possibility was further debated by Levy and Christoff (1983). More recently, Lopez-Barea and his coworkers have demonstrated that the glucose-6-phosphate dehydrogenase from Sacch. cerevisiae is inhibited by low concentrations of NADPH in cell-free extracts and that this inhibition is relieved by addition of GSSG in the presence of GSH reductase (Llobell et al., 1988). It follows that low intracellular levels of NADPH might inactivate GSH reductase in the absence of GSSG and subsequently decrease the glucose metabolism via the hexose mono- phosphate pathway (Reed, 1986). The physiological GSH-GSSG ratio should, however, provide sufficient GSSG at this level to permit retention of a significant GSH reductase activity by preventing total inactivation (Lopez-Barea and Lee, 1979). When the intracellular content of GSSG increases (e.g. under an oxidative stress), GSH reductase is reactivated and catalytic reduction of GSH is able to lower the cellular NADPH to levels that relieve inhibition of glucose-6-phosphate dehydrogenase by NADPH. As described below, reducing equivalents contained in NADPH and GSH can provide a very dynamic response during drug bioreduction processes. Arguments supporting this view were obtained in experiments with cells submitted to an oxidative challenge (Reed, 1986; Elskens and Penninckx, 1986).

2. Modulation of Radiation and Chemical Sensitivity

Manipulation of the intracellular GSH content (or GSH-dependent enzyme systems) drastically modulates the toxicity of numerous chemicals. This provides strong biological evidence that GSH is responsible for protection against or activation of these compounds. Conclusions concerning the mechanism of toxicity cannot, however, be drawn without additional investigations correlating biological data with chemical studies in a highly integrated research effort (Smith et al., 1983).

Bioreduction in activation of drugs appears to incur potential hazards for prodrugs. Prodrugs undergo conversion to active drugs essentially by two mechanisms. These are either by direct conversion or formation of an instable intermediate that undergoes a usually spontaneous reaction to yield the drug (Gorrod, 1980). These processes are not exclusive, and the intermediates may have potential for greater toxicity than the drug itself. It is apparent that a major protective role against reactive drug intermediates, generating reactive oxygen species, is provided by the

278 M J PENNINCKX AND M 7 ELSKENS

ubiquitous GSH redox cycle (Kappus, 1986; Reed, 1986). This cycle utilizes NADPH in the mitochondria1 matrix, as well as in the cytoplasm, to provide a recycled supply of GSH by GSH reductase-catalysed reduction of GSSG. However, as previously described in Section TV. C, these processes involving GSH peroxidase activity are far from being universal in micro-organisms, and in many organisms remain to be demonstrated. Examples of drugs that induce superoxide formation are the pesticides paraquat and diquat, and aromatic and heterocyclic nitro compounds (Hewick, 1982; Mason and Josephy, 1985; Reed, 1986).

The GSH redox cycle can also directly sustain bioreduction of thiol- oxidizing agents. Diazenecarbonyl derivatives were found to oxidize intracellular GSH rapidly by a well-defined set of chemical reactions (Kosower and Kanety-Londner, 1976; Kosower and Kosower, 1978). There is generally a simple stoichiometric relationship between GSH lost and the concentration of added reagent. These reactions can result in either activation or inactivation of the compound, according to the reactivity of the drug intermediate. For example, phenyldiazenecarboxylate (azo-ester) was found to oxidize GSH to GSSG on a minute scale. Excess reagent leads, however, to formation of free radicals by hydrolysis, decarboxylation and reaction of the phenyldiazene intermediate with oxygen. On the contrary, diazenedicarboxylic acid bis(N,N-dimethylamide) (diamide) was shown to penetrate cells rapidly and oxidize intracellular GSH within seconds or less at room temperature. Diamide is less susceptible to hydrolysis and quite stable in aqueous solution. Its reduction product is a stable, relatively non-toxic hydrazide. The reaction course is thus similar to that of azo-ester, but does not give rise to free radicals. Possibly, this pathway could be a protection against oxidation of cellular proteins. Nevertheless, it should be pointed out that the situation is often more complex. For example, the fungicide tetramethylthiuram disulphide (thiram), was shown to oxidize GSH in vivo and in vitro by two-step chemical reactions whose kinetics are discussed by Elskens et al. (1988a,b). For thiram concentrations below the minimal inhibitory concentration, sustained production of high levels of GSH-reducing equivalents was shown in a wild-type strain of Sacch. cerevisiae. The rates of thiram elimination displayed elements of saturation kinetics and were, in turn, regulated by the intracellular content of GSH, the ability of the yeast to provide NADPH and the specific activity of GSH reductase. Because the sensitivity to thiram was greatly enhanced in GSH-deficient mutants and in cells artificially depleted in GSH by pharmacological manipulation, bioreduction of thiram was initially thought to be involved in protection against the oxidant properties of the compound (Elskens and Penninckx, 1986). However, several lines of evidence indicated that dimethyldithiocarbamate (DMDT),

GLU rATHIONE IN MICRO-ORGANISMS 279

the reduction product of thiram, and the analogue diethyldithiocarbamate (DEDT), can undergo a variety of chemical reactions (Kumar et al., 1986). In the first place, the compounds can be re-oxidized by interaction with components of the respiratory chain, thus initiating under aerobic conditions a futile redox cycle mediated by GSH. Evidence of such processes in vitro was obtained by Kumar and his colleagues (1986). In vivo, the oxidative challenge induced by thiram in the yeast was found to decrease uptake of oxygen by galactose-induced cells, and interaction with the respiratory chain was demonstrated by in situ analysis of the redox state of electron carriers (M. T. Elskens and M. J. Penninckx, unpublished results). Secondly, oxidation of DMDT or DEDT by peroxides, and its reversal by GSH, indicated that the drugs might engage a GSH peroxidase mimic in a cyclic reaction (Kumar et al., 1986). Indeed, if the compounds might substitute GSH peroxidase in detoxifying peroxides in vivo, the GSH redox cycle would, therefore, play a significant role in chemical modification of radiation sensitivity. The mechanism of radioprotection and chemo- protection by DMDT or DEDT may have common elements with other sulphydryl radioprotection systems (Evans, 1985). On the other hand, dithiocarbamates were also found to inhibit superoxide dismutase activity because of copper-ion chelation (Heikkila et al., 1976). This effect may explain their radiosensitizing properties (Evans, 1985) as well as their role in potentiation of toxicity of drugs generating superoxide radical anions. In this connection, it would not be very surprising to find both radioprotection and radiosensitization by dithiocarbamates within the same organism. To what extent these compounds might contribute to the reaction scheme proposed is probably determined by the physiological status of the cell. For example, it appears that, in yeast, the intrinsic toxicity of thiram is modulated by the GSH redox cycle, as shown by experiments with GSH- deficient strains, but this toxicity may also vary according to the ability of yeast to grow either anaerobically or aerobically.

As already stated, GSH peroxidase mimics have been detected in cell- free extracts of two unicellular marine algae (Gennity et al., 198%). This non-enzymic activity, mediated by GSH, has been attributed to the presence of endogenous compounds such as selenocystine, selenomethionine or dimethylselenide. It was hypothesized that these compounds could enhance the antioxidant defence of algae when cells were cultivated in a selenite-containing medium. Therefore, the GSH redox cycle might be therapeutically useful in protecting against oxidative damage and have a general cytoprotective role (Halliwell, 1991). Ebselen (2-phenyl-l,2- benzisoselenazolin-3(2h)-one) is an organoselenium compound which has been developed as an antioxidant in disease therapy (Halliwell, 1991). It catalyses removal of peroxides by a cyclic reaction with intracellular GSH

280 M J PENNINCKX AND M T ELSKENS

and was shown to have anti-inflammatory effects in a number of small mammals.

Inactivation of GSH reductase has been used in several studies to assess the key importance of the GSH redox cycle during oxidant stress generated by intracellular bioreductive processes. Amongst the best known inhibitors of GSH are the nitrosourea compounds, such as 1,3-bis(2-chIoroethyl)-l- nitrosourea (BCNU) and l-(2-chloroethyl)-3-(2-hydroxyethyl)-l-nitrosourea (HeCNU). Studies by Babson and Reed (1978) indicated that inactivation of the reductase occurs only when the enzyme is in the reduced state (EH2). Inactivation appears to involve a thiocarbamate adduct, presumably with the distal thiol group of the active site. The fungicide thiram was also found to inactivate the enzyme from Sacch. cerevisiae in vivo after incubation of cells with the pesticide, in vitro in cell homogenates or with a purified enzyme preparation. The mode of action is probably similar to BCNU and also involved the reduced form of the enzyme (M. T . Elskens and M. J. Penninckx, unpublished results). Thiram was, however, not a selective inhibitor and exhibits, as already described, a variety of pharmacological activities. Nitrofurans were other compounds shown to inhibit the GSH reductase from baker’s yeast by acting as non-competitive inhibitors for NADPH and GSH. The quinone-substituted nitrofurans were found to be the most effective inhibitors (Cenas et a f . , 1991).

A search for selective inhibitors of GSH reductase is proving useful in attempts to improve current treatments against some human parasites. For example, since malarial parasites are believed to be more susceptible to oxidative stress than their host, BCNU and HeCNU were found to be efficient in preventing growth of Pf. falciparum in the early and late intra- erythrocytic stages (Zhang et a f . , 1987). Similarly, the flavin analogue 10- (4’-chlorophenyl)-3-methylflavin, which inhibits the antioxidant reductase from human erythrocytes by acting as a competitive inhibitor for GSSG, was shown to have antimalarial activity (Becker et a f . , 1990). In trypanosomatids, more effective and selective inhibitors of trypanothione reductase were recently obtained by substituting the nitrofuran moiety of nifurtimox, an existing trypanocidal drug with a relatively broad-activity spectrum (Henderson et al., 1988). These compounds were shown to undergo futile redox cycling by trypanothione reductase, and were thought to kill trypanosomes by subverting the normal antioxidant role of the enzyme (Fairlamb, 1990).

The evidence is overwhelming that the GSH redox cycle has a vital role in the cellular response to bioreduction and activation of various classes of compounds. In many instances, it is only after inactivation of GSH reductase that it is possible to observe the degree of cell injury induced by the absence of this protective system.

GLUTATHIONE IN MICRO-ORGANISMS 28 1

V. Conjugation of Glutathione: Glutathione S-Transferases

A. OCCURRENCE AND DISTRIBUTION IN MICRO-ORGANISMS

Glutathione S-transferases (EC 2.5.1.18; GSH + RX + GSR + X) were identified in 1961 and have been extensively studied since then. Numerous reviews of these enzymes have been published (Boyland and Chasseaud, 1969; Jacoby, 1978; Mannervik, 1985; Mannervik and Danielson, 1988; Boyer, 1989; Pickett et al., 1989; Waxman, 1990; van Bladeren and van Ommen, 1991) and one can estimate that the number of research articles on the enzyme, or quoting it, most likely exceeds 4000. Because of its assumed central role in biotransformation of xenobiotics, this group of enzymes has attracted much attention and new isoenzyme species are still being described. Multiple forms of GSH S-transferases may occur in living cells and the establishment of such multiplicity was, in most instances, based on chromatographic and electrophoretic separations, combined with activity measurements. For example, six major GSH S-transferases were characterized in rat liver (Jacoby et al., 1984; Mannervik, 1985). In animal tissues, the isoenzymes appear as dimeric proteins, composed of four different subunits, so that homodimers and heterodimers can exist. The enzyme was named on the basis of its constituent subunits (Jacoby et al., 1984; Mannervik, 1985; Boyer, 1989).

In reactions catalysed by the transferase isoenzymes, the sulphur atom of GSH provides electrons for nucleophilic attack on or reduction of the second electrophilic substrate. The GSH conjugate thus formed is further metabolized, and residues excreted by a well-defined sequence of reactions, the best known of which is the mercapturic pathway in animal tissues (Mannervik, 1982, 1985; Mannervik et al., 1983).

The first survey of microbial GSH S-transferases appeared at the beginning of the 1980s (Lau et al., 1980) and the enzyme was detected in numerous micro-organisms, including bacteria, protozoa, algae and fungi. Since then, several reports have been published on the occurrence and distribution of the isoenzymes in prokaryotes and microbial eukaryotes, but most of them deal principally with their evolutionary and structural relationships, rather than with their physiological functions.

Glutathione S-transferases were purified from and characterized in several members of the Enterobacteriaceae (Di Ilio et al., 1988,1991; Izuka et al., 1989; Piccolomini et al., 1989; Arca et al., 1990), in Pseudomonas aeruginosa (Piccolomini et al., 1989; Dierickx, 1991) and in Methylobacterium organophilum (Sysoev et al., 1990). The bacterial enzymes appeared to be composed of two identical subunits ( M , 22,500) and, in some instances, in the form of isoenzymes with different isoelectric points. An N-terminal

282 M J PtNNINCKX AND M T ELSKtNS

residue sequence analysis of GSH S-transferases from Proteus mirubilis showed no obvious homology with the sequence of a-, 7c- and p-classes of the mammalian enzymes or of those of plants (Di Ilio et af., 1989).

Low levels of GSH transferase activity were detected in Succharomyces cerevisiae (Shishido, 1981; Jaspers and Penninckx, 1982), but further studies have revealed the presence of substantial amounts of the enzyme in other yeast species (Casalone et al., 1988; Kuniagai et al., 1988). The enzyme was also purified from Mucor japonicus (Ando et al., 1988), Issatchenkia orientalis (Tamaki et al., 1989) and several protozoa species (Yawetz and Agosin, 1981; Overbaugh et al., 1988; Dierickx et al., 1990). Unfortunately, only very scarce information is currently available on the molecular characteristics of the transferases from microbial eukaryotes. The first reported purified microbial enzyme was for the epimastigotes of Trypanosoma cruzi, the agent of Chagas’ disease (Yawetz and Agosin, 1981). The enzyme appeared as a heterodimer with subunits of molecular weights 20,000 and 17,000, and was apparently related to the animal B forms. The enzyme from M . japonicus was shown to be made up of two identical subunits ( M , 22,000), as measured by SDS-PAGE (Ando et al., 1988), and only one major form, with a molecular weight of 35,000 estimated by gel filtration and of 33,000 by SDS-PAGE, appears to occur in Tetrahyrnena therrnophila (Overbaugh et al., 1988).

B. SUBSTRATES AND PHYSIOLOGICAL FUNCrIONS

Comprehensive descriptions of the various compounds that can serve as substrates for GSH S-transferases have been published (Chasseaud, 1979; Jacoby and Habig, 1980). An important question is whether there are endogenous substrates for GSH S-transferases in the organism or if the function of the enzymes is to detoxify xenobiotics. Later, some examples will be given of substrates that are known to arise in the metabolism of endogenous, rather than exogenous, cornpounds. Epoxides constitute a group of possible substrates that have received considerable attention. It is well established that endogenous compounds, as well as xenobiotics, may form epoxides and that GSH conjugation is a significant route in their biotransformation (Mannervik, 1985). Another group of substrates that may arise in metabolism are sulphate esters. It has been demonstrated that arylalkyl sulphates, such as benzyl sulphate, are substrates for GSI-I S- transferases. Such substrates may arise by oxidation of an alkyl group followed by sulphation (Mannervik, 1985). As stated earlier, a possible significant biological function for GSH S-transferases is protection of cell membranes against lipid peroxidation. Aldehydes, such as 4-hydroxyalkenals and malonic dialdehyde produced by lipid peroxidation,

GLUTAIHIONE IN MICRO-ORGANISMS 283

were shown to give rise to GSH conjugates. In addition, it should be noted that, in animal tissues, certain transferase isoenzymes were also characterized as species-binding steroids, bilibirubins and azo-dyes, and they appear to be involved in biosynthesis of a number of important arachidonic acid metabolites, such as prostaglandins and leukotrienes (Mannervik, 1985; van Bladeren and van Ommen, 1991).

If these findings logically suggest that GSH S-transferases may be specific enzymes designed for endogenous substrates with defined biological functions, the question about their primary cellular role still remains unanswered. In view of the fact that the individual isoenzymes demonstrate differential though overlapping substrate selectivities, the extent to which biotransformation occurs is dependent on the profile of the isoenzyme present. Consequently, both genetic and external factors causing changes in the level or activities of individual isoenzymes are of relevance with respect to the individual susceptibility towards electrophilic compounds. In many instances, cells were able to modulate their intracellular level of transferases in response to natural and artificial perturbations (Mannervik, 1985; Pickett, 1989; Vos and van Bladeren, 1990). This adaptation mechanism may thus be responsible for the acquired (de n o w ? ) role of GSH S-transferases in drug resistance. In this context, one could expect to have multiple forms of such an enzyme with a broad specificity to accommodate different types of potentially toxic agents as substrates. As described in Section 11, GSH in trypanosomatids is mainly in the form of spermidine conjugates. This contribution does not, for instance, rule out a possible involvement of free GSH in cellular detoxification of protozoa. Recent investigations, focusing on the effect of intracellular free GSH on the susceptibility of T. cruzi to trypanocidal drugs such as nifurtimox and benzomidazole, have shown a positive correlation between GSH content and resistance to drugs (Moncada et al., 1989). Furthermore, BSO which was used to lower the intracellular level of GSH in T. cruzi, was found to potentiate the toxicity of both drugs. The implication of GSH S-transferases in the mechanism of resistance of Te. thermophila and Beweria sp. to isosorbide dinitrate was recently discussed (Ropenga and Lenfant, 1987; Ropenga et al., 1989). It was shown that the compound induced transferase activity and that this activity correlated well with the rate of drug bioconversion into the 5- and 2-mononitrate forms.

The organochlorine pesticide captan was shown to inhibit growth of the non-symbiotic nitrogen-fixing bacterium Azospirillum brasilense. A mutant that contained high levels of GSH and GSH S-transferases was isolated and found to be resistant to the pesticide (Gallori et al., 1988). Similarly, captan-resistant strains of Botrytis cinerea were apparently able to regulate their biosynthesis of GSH and GSH S-transferase activity when grown in the presence of the pesticide (Barak and Edgington, 1984).

284 M. J. PENNINCKX A N D M T ELSKENS

The plasmid conferring resistance to fosfomycin in Escherichia coli was shown to encode for a GSH S-transferase protein (Arca et al., 1988). Mutants deficient in GSH were more susceptible to the antibiotic, indicating that the tripeptide is crucial for the detoxification pathway. The enzyme which mediates resistance was further purified and characterized as a typical GSH S-transferase, catalysing opening of the epoxide ring of the antibiotic to form an inactive adduct (Arca et al., 1990).

In Salmonella typhimurium which is a tester strain for the Ames test, GSH and the transferase isoenzymes were identified as possible factors affecting mutagenicity of xenobiotics (Summer et al., 1980). Possible roles for GSH and the transferase system were also reported in degradation of methidathion by Bacillus coagulans (Gauthier et al., 1988), in the resistance of Aspergillusfiavus to endogenous aflotoxin (Saxena et al., 1988), and in detoxication of aromatic xenobiotics by Cunninghamella elegans (Wackett and Gibson, 1982) and by a Fusarium sp. (Cohen et al., 1986).

Although these data illustrate the role played by GSH and the transferase isoenzymes in detoxification of or resistance to several xenobiotics in micro- organisms, it should be stressed that, in a number of micro-organisms, biosynthesis and biotransformation of GSH S-conjugates lead to formation of toxic metabolites (Kerklaan et al., 1985; Anders, 1988; Anders et al., 1988). For example, bacterial GSH might activate numerous mutagens, including l-chloro-2,4-dinitrobenzene, substituted nitrosoguanidines, styrene-7,8-oxide, 1,2-dibromoethane, methyl methane sulphonate and, quite possibly, halogenated alkenes.

VI. Other Aspects of Glutathione Function

A. THE GLYOXALASE PATHWAY

Hydration and rearrangement of methylglyoxal(2-oxopropanal) to D-lactic acid are catalysed by a GSH-dependent system, termed glyoxalase, consisting of two distinct enzymes (Racker, 1951), namely glyoxalase I (lactoylglutathione lyase; EC 4.4.1.5) and glyoxalase I1 (hydroxyacyl- glutathione hydrolase; E C 3.1.2.6). The reactions involved are:

methylglyoxal + GSH hemithioacetal (non-enzymic)

hemithioacetal + S-D-lactoylglutathione (glyoxalase I)

(glyoxalase 11) S-D-lactoylglutathione + H20 + D-lactic acid + GSH

Despite numerous efforts, the biological function of the system remains puzzling, especially in micro-organisms. Recent advances in this field with

GLUTATHIONE IN MICRO-ORGANISMS 285

animals and plants have, however, suggested that the glyoxalase system may be associated with regulation of cell proliferation and maturation, vesicle mobilization and disease processes, such as tumour growth and diabetes mellitus (Thornalley, 1990). Glyoxalases were initially thought to be involved on a major metabolic pathway for breakdown of glucose to D-

lactate via methylglyoxal (Harden, 1932). However, the discovery of phosphorylated glycolytic intermediates, and the finding that L-lactate was the major product of glycolysis in animal tissues, have disclaimed the glyoxalase system as belonging to mainstream hexose catabolism.

Glyoxalase I was studied mainly in animal tissues and Saccharomyces cerevisiae, but was also identified in prokaryotes (Thornalley, 1990). Unlike the mammalian enzyme, which is a homodimer, microbial glyoxalases I exist in the form of monomeric entities with a fairly broad substrate specificity towards a-ketoaldehydes; they have high in vitro K , values for the hemithioacetal adduct (Rhee et al., 1986; Douglas et al., 1986). The encoding genes for the enzymes from Sacch. cerevisiae and Pseudonzonas putida have been cloned and characterized (Rhee et al., 1988). Several lines of evidence indicate that the origin of bacterial glyoxalase I might be essentially different from that of its eukaryotic counterpart.

Much less is known about glyoxalase 11, although the enzyme has been purified from Sacch. cerevisiae (Murata et al., 1986b). Determination of its molecular weight by gel filtration and SDS-PAGE gave a value of about 19,000, which is within the range observed for mammalian enzymes (Thornalley 1990). Amongst several thiol esters tested, the yeast enzyme appeared quite specific and hydrolysed only S-lactoylglutathione with a K,,, value of 7 PM. This value is extraordinarily low in comparison to those measured for mammalian enzymes, which ranged between 180 and 440 PM. It should be pointed out that hydrolysis of S-lactoylglutathione is also shared in Sacch. cerevisiae by a GSH thiol esterase (Murata et al., 1987). Nevertheless, after purification, this protein appears totally distinct from glyoxalase 11.

Early research work on the physiological role of the glyoxalase pathway logically first focused on mechanisms of methylglyoxal production and its susequent fate. A reaction sequence providing a by-pass to the glycolytic pathway was discovered in Escherichia coli by Cooper and his coworkers (Cooper and Anderson, 1970). This sequence of reactions, which is a combination of phosphorylated and non-phosphorylated pathways for breakdown of glucose, involves a methylglyoxal synthase activity and the GSH-dependent glyoxalase system. However, the physiological function of the by-pass is still a matter for conjecture. Methylglyoxal synthase and the glyoxalase system in E. coli belong to a constitutive pathway. At the beginning, it was suggested that this pathway could be operative under

286 M J PENNINCKY AND M T F l SKENS

phosphate limitation, a situation that impairs action of glyceraldehyde-3- phosphate dehydrogenase and prevails often under ecological conditions (Cooper, 1984). Since methylglyoxal synthase is strongly inhibited by inorganic phosphate in vitro, limitation of inorganic phosphate could allow a significant metabolic carbon flow through the non-phosphorylated pathway. Unfortunately, no experimental evidence has been presented to support this hypothesis. Another interesting, but related, supposition is that an operative glyoxalase by-pass would permit dissociation of glucose breakdown from synthesis of ATP (Cooper, 1984). In effect catabolism of dihydroxyacetone phosphate, which is mediated by methylglyoxal synthase, might provide the inorganic phosphate required to trigger the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase that leads to phosphoenolpyruvate biosynthesis. It is noteworthy that resting cells of E. coli, energetically disconnected, still utilize about 50% of the glucose used in the medium (Roberts et al . , 1963). Methylglyoxal has also been proposed to serve as a precursor of D-lactate, which can be subsequently used to energize transport systems for sugars and amino acids in E. coli (Kaback, 1974).

Despite all of these hypotheses, it is obvious that the glyoxalase pathway in E. coli is still enigmatic and apparently inadequate for channelling of large amounts of 2-oxoaldehydes (Hopper and Cooper, 1971). Indeed, challenging cells fully derepressed for glycerol catabolism and having lost feedback control on glycerol kinase with fructose 1,6-bisphosphate resulted in production of lethal amounts of methylglyoxal. Resistance to methylglyoxal in E. coli can, however, be achieved by a mutational or a genetically engineered increase in the intracellular content of GSH and the level of glyoxalase activities (Murata et al., 1980; Murata and Kimura, 1990).

In a Pseudomonas sp, it was suggested that gluconate metabolism may proceed by a glyoxalase by-pass which involves glyceraldehyde 3-phosphatase and glyceraldehyde dehydratase catalysing formation of methylglyoxal (Rizza and Hu, 1973). Arguments supporting this view were supplied using differently labelled gluconate substrates. The proposed pathway, at variance with the classical Entner-Doudoroff scheme, implies that substrate-level phosphorylation may not occur in conversion of gluconate to pyruvate and could account for the strictly aerobic life-style of the pseudomonad.

The status of the glyoxalase pathway in Sacch. cerevisiae appears completely different. More precisely, enzyme production seems to be regulated. For example, growth on glycerol as the carbon source or on glucose in the presence of methylglyoxal induced glyoxalase activities. The system was also shown to respond to the glucose effect (Penninckx et al.,

G I LITATHIONE IN MICRO-ORGANISMS 287

1983). Furthermore, it was demonstrated that the activity of the glyoxalase system is related to the overall growth status of Sacch. cerevisiae (Dudani et al., 1984). High levels of activity were detected in actively growing cells, whereas low levels were expressed in resting cells. It should be noted that a similar phenomenon was encountered in plants (Ramaswamy etal., 1983). Interestingly, this observation raises the question of the existence of a constitutive methylglyoxal production in Sacch. cerevisiae and recalls an old hypothesis put forward by Szent-Gyorgyi and his coworkers (Egyud and Szent-Gyorgy, 1968). These authors suggested that methylglyoxal acted as a physiological growth inhibitor whose effect might be relieved by the glyoxalase system, growth resulting from the balance between both effects. In this connection, certain yeast species which dissimilate methanol were shown to have a complete and constitutive glycolytic by-pass similar to that of E. coli (Babel and Hofrnan, 1981).

A mutant fully defective glyoxalase I , bearing only one nuclear mutation, has been isolated from Sacch. cerevisiae (Penninckx et al., 1983). This strain, which is killed by exposure to glycerol, was found to accumulate about 10 times more intracellular methylglyoxal than the wild type when cells were transferred from a glucose-containing medium to a glycerol- containing medium. Since methylglyoxal synthase activity was not detected in the strain, it was suggested that the oxoaldehyde formed was derived from spontaneous decay of intracellular glyceraldehyde 3-phosphate which accumulates during growth on glycerol. Therefore, glyoxalase I could play a leading role in detoxification of methylglyoxal that accumulates as a consequence of non-regulated glycerol catabolism. In essence, the system shows some resemblance to formation of catalase and superoxide dismutase which function in a similar relationship to oxidative metabolism. Glyoxalase I from yeast is not absolutely specific for methylglyoxal and may utilize numerous 2-oxoaldehydes. In this connection, other possible sources of oxoaldehyde in yeast might be aminoacetone and 1 -hydroxyacetone formed during catabolism of threonine, isoleucine and valine (Murata et a l . , 1986a,b).

To conclude, it could be said that, in spite of progress in understanding the nature of the reactions catalysed by the glyoxalase system, the role and fate of methylglyoxal in living cells remain largely unexplained. Current views suggest that the glyoxalase pathway can function as a possible detoxification pathway for endogenous oxoaldehydes or in intermediary metabolism. Recent observations in this field with mammalian cells have focused attention on the multiple effects of S-D-lactoylglutathione, includ- ing potential therapeutic uses (Gillespie, 1979; Thornalley, 1990). Genetically engineered cells of E. coli, which carry the gene (glol) encoding for glyoxalase I biosynthesis in P. putida, were suggested as a useful

288 M. 1. PENNINCKX AND M. T. ELSKENS

tool for commercial production of S-D-lactoylglutathione (Murata and Kimura, 1990).

B . METHANOL DISSIMILATION

Methylotrophs perform their total cellular biosynthesis from C1 com- pounds. Diversity in metabolic strategies for C, compounds, and particularly methanol catabolism, have been extensively described for bacteria (Anthony, 1982; Sysoev et al., 1990). By contrast, quite homo- geneous pathways were observed among microbial eukaryotes, especially in methylotrophic yeasts, with respect to the mechanism of methanol dissimilation and assimilation (Sahm, 1977; van Dijken et al., 1981). When grown on methanol, Candida boidinii and Hansenula polymorpha synthesized crystalline cytoplasmic peroxisome inclusions containing methanol oxidase and catalases (Fukui and Tanaka, 1979). Formaldehyde, produced during catabolism of methanol, is exported to the cytosol, where it apparently reacts spontaneously with GSH to form S-hydroxylmethyl- glutathione, a hemimercaptal adduct. The NADf-linked formaldehyde dehydrogenase (EC 1.2.1.1) catalyses subsequent biotransformation of the compound into S-formylglutathione, which is rapidly hydrolysed to formate and GSH by a separate enzyme, S-formylglutathione hydrolase (EC 3.1.2.12). Formate is then oxidized to give carbon dioxide by a second dehydrogenase system (Fig. 6).

I 0 2 OXIDASE

C H @ H ~ s H C M

H20 GHZo2 1R 0 2

I'EROXISOME i

FORMALDEHYDE S-FORMYIGLLITATHIONE DEHYDROGENASE HYDROIASE

G9I4 * G s H \ H

CYTOSOL

FIG. 6. Pathway describing oxidation of methanol to carbon dioxide in methylotrophic yeasts.

Formaldehyde dehydrogenase is as ubiquitous as glyoxalase in living cells (Uotila and Koivusalo, 1983). The enzyme was detected and partially purified 30 years ago from baker's yeast (Rose and Racker, 1962) and was snown to be present in considerable amounts in methanol-utilizing yeasts

GLUTATHIONF IN MICRO-ORGANISMS 289

(Sahm, 1977; Egli et al., 1980). Several but not all methylotrophic bacteria contain the enzyme, which is also found in E. coli (Cox and Quayle, 1975; Ben-Bassat and Goldberg, 1977). Formaldehyde dehydrogenase from C . boidinii was shown to be a dimer consisting of two identical subunits with a molecular weight of about 40,000 (Schiitte et al., 1976). This value is similar to those reported f m mammalian enzymes (Uotila and Koivusalo, 1974).

S-Formylglutathione hydrolase, which catalyses irreversible hydrolysis of S-formylglutathione, was originally discovered and purified to homo- geneity from human liver (Uotila and Koivusolo, 1983). Homogeneous preparations have also been described for the methylotrophic yeasts Kloeckera sp. and C. boidinii (Kato et al. , 1980; Neben et al., 1980). The enzyme from these yeasts appears as a heterodimer with a molecular weight of about 60,000 and is highly specific for S-formylglutathione (van Dij ken et al . , 1976).

Formaldehyde dehydrogenase and S-formylglutathione hydrolase parti- cipate in metabolism of methylotrophic bacteria and yeasts, on the pathway for complete oxidation of methanol to carbon dioxide (Sahm, 1977). Formaldehyde, S-formylglutathione and formate are intermediates on this pathway, which provides energy for growth. In many micro-organisms, synthesis of formaldehyde dehydrogenase is apparently controlled by derepression-repression processes (Sahm, 1977; Neben et al., 1980). In non-methylotrophic organisms, such as Sacch. cerevisiae and E. coli, it is quite possible, however, that formaldehyde dehydrogenase has a role in detoxifying formaldehyde. Substantial evidence supporting this view was reported for animal tissues (Uotila and Koivusalo, 1983). Formaldehyde can be formed on several metabolic pathways, such as catabolism of methionine and choline, oxidation of methanol by an alcohol dehydrogenase and catalase or by other minor metabolic reactions.

Methylglyoxal is a good substrate for formaldehyde dehydrogenase. Utilization of methylglyoxal by the action of glyoxalase or formaldehyde dehydrogenase is thus dependent on GSH and results in GSH thiol-ester iormation. Therefore, besides functioning in detoxification of formaldehyde and methylglyoxal, formaldehyde dehydrogenase may have some functions associated with thiol-ester products. As already indicated, specific GSH esterases that catalyse hydrolysis of the products have a!so been demonstrated in yeasts (Murata et al., 1987).

C. HEAVY-METAL DETOXIFICATION

A higher sensitivity of GSH-deficient mutants of E. coli towards heavy metals has been reported (Apontoweil and Berends, 1975b). On a chemical

290 M J PI-NNINCKX AND M T ELSKENS

basis, it was previously expected that GSH would be able to form chelation complexes with heavy metals and may, in this way, be involved in a detoxification process. Recent progress in this field was obtained by studies with Schizosaccharornyces pombe and Candida (Torulopsis) glabrata (Grill et al., 1985; Dameron et al., 1989). When exposed to cadmium salts, these yeasts were shown to synthesize peptide derivatives that involved cadmium ions and multiple moieties of GSH (Grill et al., 1985; Hayashi et al., 1988; Mehra et al . , 1988). The so-called cadystins or phytochelatins (Grill et al., 1987) were apparently also found in some plant species (Jackson et al., 1987), but remained undetected in bacteria, Sacch. cerevisiae and animal tissues.

It was further shown that the pathway for heavy-metal inactivation was apparently dependent on growth conditions. When cultivated in nutrient broth, Schiz. pombe and T. glabrata, exposed to cadmium salts, form cadmium sulphide particles coated with GSH and y-glutamylcysteine whereas when grown in minimal media the cadmium adduct was coated with peptides having the general structure (y-Glu-Cys),-Gly (Dameron et al., 1989). Two pathways for biosynthesis of this compound were found in cell-free extracts of Schiz. pombe (Hayashi et al., 1991). The first involves a transfer of y-Glu-Cys from both GSH and cadystins to GSH and cadystins, whereas the second is a polymerization of y-Glu-Cys from (y-Glu-Cys), and GSH to give (y-Glu-Cys),+,, followed by addition of a glycine residue catalysed by GSH synthetase. Mutants of Schiz. pombe deficient in GSH were recently described (Glaeser et al., 1991). The Gsh- mutants have lost the ability to excrete cadmium and were also shown to be more susceptible to other heavy metals like bismuth, copper, lead, zinc and silver.

VII. Concluding Remarks

It is now clearly established that GSH and related compounds are widespread in the microbial world, especially amongst organisms with an aerobic life-style. This observation emphasizes the role of GSH in cellular protection against by-products generated by oxidative metabolism, but it does not in any way limit its functions to this role. Glutathione has, indeed, been shown to act as an enzyme cofactor, transport component, nucleophilic substrate and sulphur reservoir, and it also participates in key cellular processes such as protein synthesis and degradation, regulation of enzyme activity, synthesis of DNA, and maintenance of the integrity of cell membranes and organelles. Having such functional diversity, GSH is interrelated with a number of metabolic pathways and its intracellular modulation could obviously have an impact on the entire cell, making it

GLUTATHIONE IN MICRO-ORGANISMS 29 1

extremely difficult to associate directly a given cellular end-point with one molecule or system. Nevertheless, genetic or pharmacological manipulations that alter GSH status were found to be useful for investigating the role of the tripeptide in detoxification of xenobiotics and its function as a scavenger of externally and possibly internally formed radicals. One might be astonished to note that, both in Escherichia coli and Saccharomyces cerevisiae, GSH appears to play an important role in cellular protection during chemical stresses in spite of the fact that key enzymes of detoxifica- tion, such as GSH peroxidase and GSH S-transferase, remain at a low level. This protective effect could be attributed to the GSH redox cycle and also highlights the chemical reactivity of the tripeptide.

It is appropriate to mention the profound influence exerted by investiga- tions of Meister and the Kosowers on the early development of microbial GSH research. Since then, further knowledge has been gained on the specific metabolism and functions of GSH in micro-organisms, and mammalian physiologists take an ever greater interest in the use of microbial models for understanding peculiar aspects of metabolism in animal tissues. It is expected that, in the future, the GSH network will be extended in all possible directions.

VIII. Acknowledgements

This work was supported in part by research grants from the Fonds National de la Recherche Scientifique (FNRS) to M. J. P., and an Actionde Recherche Concertee (ARC) financed by the Belgian State. The skilful assistance of Anne Wies and Fernand-Pierre Wies was very much appreciated.

REFERENCES

Abdrashitova, S. A., Mynbaeva, B. N., Aidarkhanov, B. B. and Ilyaletdinov, A. N. (1990).

Aisaka, K., Uwajima, T. and Tereda, 0. (1983). Agricultural and Biological Chemistry 47,

Allen, L., Meck, R. and Yunis, A. (1980). Research Communications in Chemical Pathology

Anders, M. W., Lash, L., Dekant, W., Elfarra, A. A. and Dohn, D. R. (1988). Critical

Ando, K., Honma, M., Chiba, S., Tahara, S. and Mizutani, J . (1988). Agricultural and

Anfinsen, C . B. (1973). Science 181, 223. Ansorge, S. , Bohley, P., Kirschke, H., Langgner, J., Marquardt, L., Wiederanders, B. and

Ansorge, S., Bohley, P., Kirschke, H. , Langger, J., Wiederanders, B. and Hanson, H.

Anthony, C. (1982). In “The Biochemistry of Methylotrophs”. Academic Press, London.

Mikrobiologiya 59, 234.

1269.

and Pharmacology 21, 175.

Reviews in Toxicology 18, 31 1.

Biological Chemistry 52, 135.

Hanson, H. (1973a). FEBS Letters 37, 238.

(197313). European Journal of Biochemistry 32, 27.

292 M. J . PENNINCKX AND M. T. ELSKENS

Apontoweil, P. and Berends, W. (1975a). Biochimica et Biophysica Acta 399. 1. Apontoweil, P. and Berends, W. (1957b). Biochimica et Biophysica Acta 399, 10. Arca, P . , Rico, M., Brana, A. F., Villar, C. J., Hardisson, C. and Suarez, J . E. (1988).

Arca, P., Hardisson, P. and Suarez, J. E . (1990). Antimicrobial Agents and Chemotherapy

Arias, I. M. and Jacoby, W. B. (eds) (1976). “Glutathione: Metabolism and Function” (Kroc

Babel, W. and Hofmann, K. H. (1981). FEMS Microbiology Letters 10, 133-136. Babson, J. R. and Reed, D. J . (1978). Biochemical and Biophysical Research Communications

Barak, E. and Edgington, L. V. (1984). Pesticide Biochemistry and Physiology 21, 412. Baron, E. S. (1951). Advances in Enzymology 11, 201. Becker, K., Christopherson, R. I . , Cowden, W. B., Hund, N. H. and Schirmer, R. H. (1990).

Bellofatto, V., Fairlamb, A. H., Henderson, G. B. and Cross, G. A. (1987). Molecular and

Ben Bassat, A. and Goldberg, I . (1977). Biochimica et Biophysica Acta 497, 586. Bonsignore, A. and De Flora, A. (1972). Current Topics in Cellular Regulation 6, 21. Bouter, S. , Kerklaan, P. R. M., Zoetemelk, C. E. M. and Mohn, G. R. (1988). Biochemical

Boyer, T. D. (1989). Hepatology 9, 486. Boyland, E. and Chasseaud, L. F. (1969). Advances in Enzymology 32, 173. Branden, C. I., Eklund, H. and Soderberg, B. 0. (1983). In “Functions of Glutathione:

Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 251. Raven Press, New York.

Antimicrobial Agents and Chemotherapy 32, 1552.

34, 844.

Foundation Series, vol. 6 ) . Raven Press, New York.

83, 754.

Biochemical Pharmacology 39, 59.

Biochemical Parasitology 25, 227.

Pharmacology 37, 577.

Brown, R. W. (1974). Journal of Dairy Science 57, 797. Bruinenberg, P. M., van Dijken, J. P. and Scheffers, W. A. (1983a). Journal of General

Bruinenberg, P. M., van Dijken, J. P. and Scheffers, W. A. (1983b). Journal of General

Buchanan, B. B. (1980). Annual Review of Plant Physiology 31, 314. Casalone, E. , Di Illio, C., Federici, G. and Polsinelli, M. (1988). Antonie van Leeuwenhoek

Ctnas, N. K., BironaitC, D. A , , Kulys, J. J . and Sukhova, N. M. (1991). Biochimica et

Chance, B., Sies, H. and Boveris, A. (1979). Physiological Reviews 59, 527. Chasseaud, L. F. (1979). Advances in Cancer Research 29, 175. Cheng, H., Aronson, A . and Holt, S. (1973). Journal of Bacteriology 113, 1135. Cherest, H., Eichler, F. and de Robichon-Szulmajster, H. (1969). Journal of Bacteriology

Chung, Y. C. and Hurlbert, R. E. (1975). Journal of Bacteriology 123, 855. Cohen, E., Gambiel, A. and Katan, J . (1986). Pesticide Biochemistry and Physiology 26, 1. Cohen, G. M. and Friedman, R. B. (1982). Biochemical Society Transactions 10, 78. Colowick, S . , Lazarow, A., Racker, E., Schwarz, D. R., Stadtman, E. and Waelsh, H. (eds)

Cooper, R. A. (1984). Annual Review of Microbiology 38, 49. Cooper, R. A. and Anderson, A. (1970). FEBS Letters 11, 273. Cox, R. B. and Quayle, J . R. (1975). Biochemical Journal 150, 569. Creighton, T. E. (1983). In “Functions of Glutathione: Biochemical, Physiological,

Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and €3. Mannervik eds), p. 205. Raven Press, New York.

Microbiology 129, 953.

Microbiology 129, 965.

54, 367.

Biophysica Acta 1073, 195.

97, 328.

(1954). “Glutathione”. Academic Press, London.

Crook, E. M. (1959). Biochemical Society Symposium 17, 116,. Csonka, L. N. and Clark, A. J. (1980). Journal of Bacteriology 143, 529. Dameron, C. T., Reese, R. N., Mehra, R. K., Kortan, A. R., Caroll, P. J., Steigenvald,

M. L., Brus, L. E. and Winge, D . R . (1989). Nature 338, 596.

GLUTATHIONE IN MICRO-ORGANISMS 293

Danson, M. J., Eisenthal, R., Hall, S . , Kessel, S. R. and Williams, D. L. (194X). Biochemical

de Rey-Pahlade, J. (1888). AcadCmie des Sciences (Paris) 107, 43. Dean, R. T., Thomas, S. M. and Garner, A. (1986). Biochemical Journal 240, 489. Di Ilio, C., Aceto, A, , Piccolomini, R., Allocati, N., Faraone, A, , Cellini, L., Ravagnan,

Di rho, C., Aceto, A, , Piccolomini, R., Allocati, N., Faraone, A , , Bucciarelli, T. , Barra,

Dierickx, P. J. (1991). Archives Internationales de Physiologie et de Biochimie 100, 10. Dierickx, P. J., Almar, M. M. and De Jonckheere, J. F. (1990). Biochemistry International

Douglas, K. T., Seddon, A. P. andNakagawa, Y. (1986). International Journal of Biochemistry

Drotar, A , , Burton, G., Tavernier, J. E. and Fall, R. (1987). Applied and Environmental

Dubois, E., Vissers, S . , Grenson, M. and Wiame, J.-M. (1977). Biochemical and Biophysical

Dudani, A. K., Srivastava, L. K. and Prasad, R. (1984). Biochemicaland Biophysical Research

Dupouy, D., Conter, A, , Croute, F., Murat, M. and Planel, H. (1985). Environmental and

Eddy, A. A. (1982). Advances in Microbial Physiology 23, 1. Eggleston, L. V. and Krebs, H. A. (1974). Biochemical Journal 138, 425. Egli, T. , van Dijken, J. P., Vcenhuis, M., Harder, W. and Fiechter, A. (1980). Archives of

Egyud, L. G. and Szent-Gyorgy, A. (1968). Science 160, 1140. Eklund, H., Cambillau, C., Sjoeberg, B. M., Holmgren, A, , Joernvall, H., Hoeoeg, J . 0.

Elliott, K. and Whelan, J. (eds) (1979). Ciba Foundation Symposium 72. Elskens, M. T. and Penninckx, M. J. (1986). Archives Internationales de Physiologie et de

Elskens, M. T. , Penninckx, M. J . , Vandeloise, R. and Vander Donckt, E. (1988a).

Elskens, M. T., Penninckx, M. J., Vandeloisc, R. and Vander Donckt, E. (1988b). Bulletin

Journal 218, 81 1.

G. and Federici, G. (1988). Biochemical Journal 255, 971.

D . and Federici, G. (1991). Biochimica et Biophysica Acta 1077, 1431.

22, 593.

18, 549.

Microbiology 53, 1626.

Research Communications 75, 233.

Communications 119, 962.

Experimental Botany 25, 339.

Microbiology 124, 115.

and Braenden, C. I. (1984). EMBO Journal 3, 1443.

Biochimie 94, 76.

International Journal of Chemical Kinetics 20, 837.

de la SociCtt! Chimiaue Belee 97. 397. Y ,

Elskens, M. T., Jaspers, C. J. and Penninckx, M. J. (1991). Journal of General Microbiology 137, 637.

Ermler, U. and Schulz, G. E. (1991). Proteins: Structure Function and Genetics 9,

Evans, R. G. (1985). International Journal of Radiation, Oncology, Biology, Physics 11,

Eyer, P. Bnd Podhradsky, D. (1986). Analytical Biochemistry 153, 57. Fahey, R. C. (1977). In “Protein Crosslinking, part A” (R. B. Friedman, ed.), p. 1. Plenum

Press, New York. Fahey, R. C. and Newton, G. L. (1983). In “Functions of Glutathione: Biochemical,

Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S . Orrenius, A. Holmgren and B. Mannervik, eds), p. 251. Raven Press, Ncw York.

174.

1163.

Fahey, R. C. and Newton, G. L. (1986). Methods in Enzymology 143, 85. Fahey, R. C., Brody, S. and Mikoljczyk, S . D. (1975). Journal of Bacteriology 121,

Fahey, R. C., Brown, W. C., Adams, W. B. and Worsham, M. B. (1978). Journal of

Fahey, R. C., Bushbacher, R. M. and Newton, G. L. (1987). Journal of Molecular Evolution

Fahey, R. C., Newton, G. L., Arrick, B., Overdank-Bogart, T. and Aley, S . B. (1984).

144.

Bacteriology 133, 1126.

25, 81.

Science 224. 70.

294 M. J. PENNINCKX AND M. T. ELSKFNS

Fairfield, A. S. , Abosch, A . , Ranz, A., Eaton, J. W. and Meshnick, S. R. (1988). Molecular

Fairlamb, A. H. (1989). Parasitology 99, 93. Fairlamb, A. H. (1990). Biochemical Society Transactions 18, 717. Fairlamb, A. H. and Cerami, A. (1985). Molecular and Biochemical Parasitology 14, 187. Fairlamb, A. H., Blackburn, P. , Ulrich, P., Chait, B. T. and Cerami, A. (1985). Science 227,

1485. Fairlamb, A. H., Henderson, G. B. and Cerami, A. (1986). Molecular and Biochemical

Parasitology 21, 247. Fairlamb, A. H., Henderson, G. B., Bacchi, C. J. and Cerami, A. (1987). Molecular and

Biochemical Parasitology 24, 185. Ficchter, A,, Fuhrmann, G. F. and Kappeli, 0. (1981). Advances in Microbial Physiology

22, 123. Flohe, L. and Gunzler, W. A. (1976). In “Glutathione: Metabolism and Function” (Kroc

Foundation Series, vol. 6) (I. M. Arias and W. B. Jacoby, eds), p. 17. Raven Press, New York. Flohe, L., Benohr, H. C., Sies, H., Waller, H . D. and Wendel, A . (eds) (1974).

“Glutathione”, Thieme, Stuttgart. Flohe, L., Gunzler, W. A. and Loshen, G. (1980). In “Trace Metal in Health and Disease”

(N. Khararash, ed.), p. 263. Raven Press, New York. Fox, G. E., Stakebrandt, E. , Hespell, R. B . , Gibson, J., Maniloff, J., Dyer, T. A,, Wolfe,

R. S. , Balch, W. E., Tanner, R. S., Magrum, L. J., Zablen, L. B., Blakemore, R., Gupta, R., Bonen, L., Lewis, B. J., Stahl, D. A, , Luehrsen, K. R., Chen, K. N. and Woese, C. R. (1980). Science 209, 4.57.

and Biochemical Parasitology 30, 77.

Freedman, R. B. (1979). FERS Letters 97, 201. Freedman, R. B. and Hillson, D . A. (1980). In “The Enzymology of Post-Translational

Modification of Proteins I” (R. B. Freedman and H. C. Hawkins, eds), p. 157. Academic Press, London.

Freedman, R. B., Hawkins, H. C., Murant, S . J. and Reid, L. (1988). Biochemical Society Transactions 16, 96.

Fridovich, I. (1982). Biochemical Society Transactions 10, 67. Fuchs, J. (1977). Journal of Bacteriology 129, 140. Fuchs, J. A. and Warner, H. R. (1975). Journal of Bacteriology 124, 140. Fuchs, J. A,, Hailer, B. and Tuggle, C. K. (1983). In “Functions of Glutathione: Biochemical,

Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 385. Raven Press, New York.

Fukui, S . and Tanaka, A. (1979). Trends in Biochemical Sciences 4, 246. Galiazzo, F. , Schlesser, A. and Rotilio, G. (1987). Biochemical and Biophysical Research

Gallori, E., Casalone, E., Nocita, O., Turbanti, L. and Polsinelli, M. (1988). Journal of

Gancedo, C. and Serrano, R. (1989). In “The Yeasts” (A. H. Rose and J. S. Harrison, eds),

Gauthier, M. J . , Berge, J. B., Cuany, A , , Breittmayer, V. and Fournier, D. (1988). Pesticide

Gcnnity, J . M., Bottino, N. R., Zingaro, R. A, , Wheeler, A. E. and Irgolic, K. J. (1985a).

Gennity, J. M., Bottino, N. R., Zingaro, R. A, , Whceler, A. E. and Irgolic, K. J . (198%).

Ghisla, S. and Massey, V. (1989). European Journal of Biochemistry 181, 1. Giammanco, G., Buissieres, J., Toucas, M., Brault, G. and Le Minor, L. (1980). Annales

Giddings, T., Wolk, P. and Shomer-Ilan, A. (1981). Journal of Bacteriology 146, 1067. Gillespie, E. (1979). Nature 277, 135. Giovannelli, J. (1987). Methods in Enzymology 143, 419. Glaeser, H., Coblenz, A., Kruczek, R., Ruttke, I., Ebert-Jung, A. and Wolf, K. (1991).

Communications 147, 1200.

General Microbiology 134, 3173.

2nd cdn, vol. 3, pp. 205-259. Academic Press, London.

Biochemistry and Physiology 31, 61.

Phytochernistry 24, 2817.

Phytochemistry 24, 2823.

de Microbiologie de l’lnstitut Pasteur (Paris) 131, 181.

Current Genetics 19. 207.

GLUTATHIONE IN MICRO-ORGANISMS 295

Gleason, F. K. and Holmgren, A. (1988). FEMS Microbiology Reviews 4, 271. Goldberger, R. F., Epstein, C. J . and Anfinsen, C. B. (1963). Journalof Biological Chemistry

Gorrod, J. W. (1980). Chemical Industry 7, 457. Greer, S. and Perham, R. N. (1986). Biochemistry 25, 2736. Grenson, M. and Hennaut, C. (1971). Journal of Bacteriology 105, 477. Griffith, 0. W. (1987). Methods in Enzymology 143, 366. Griffith, 0. W. and Meister, A . (1979a). Proceedings of the National Academy of Sciences

Griffith, 0. W. and Meister, A. (1979b). Journal of Biological Chemistry 254, 7558. Griffith, 0. W. and Meister, A. (1980). Proceedings of the National Academy of Sciences of

238, 628.

of the United States of America 76, 5606.

the United States of America 77, 3384. Griffith, 0. W., Anderson, M. E. and Meister, A. (1979). Journal of Biological Chemistry

254. 1205. Grill, E., Zenk, M. H. and Winnacker, E . (1985). Science 230, 674. Grill, E., Winnacker, E. L. and Zenk, M. H . (1987). Proceedings of the National Academy

Gushima, H., Miya, T., Murata, K. and Kimura, A. (1983). Journal ofApplied Biochemistry

Halliwell, B. (1991). Drugs 42, 569. Halliwell, B. and Aruoma, 0. I. (1991). FEBS Letters 281, 9. Halliwell, B. and Gutteridge, J. M. (eds) (1989). “Free Radicals in Biology and Medicine”.

Halliwell, B. and Gutteridge, J. M. (1990). Methods in Enzymology 186, I. Harden, A. (1932). In “AlcoholicFermentation”, p. 109. Longmans, Green and Co., London. Harington, C. R. and Mead, T. H. (1935). Biochemical Journal 29, 1602. Hasegawa, M. and Matsubana, I . (1978). Agricultural and Biological Chemistry 42, 385. Hayashi, Y., Nakagawa, C. W., Uyakul, D., Imai, K. , Isobe, M. and Goto, T. (1988).

Hayashi, Y . , Nakagawa, C. W., Mutoh, N., Isabc, M. and Goto, T. (1991). Biochemistry

Heikkila, R. E., Cabbat, F. S. and Cohen, G. (1976). Journal of Biological Chemistry 251,

Henderson, G. B . , Fairlamb, A. H., Ulrich, P. and Cerami, A. (1987). Biochemistry 26,3023. Henderson, G. B., Ulrich, P., Fairlamb, A. H., Rosenberg, I., Pereira, M., Sela, M. and

Cerami, A. (1988). Proceedings of the National Academy of Sciences of the United States of America 85, 5374.

Henderson, G. B., Yamachuci, M., Novoa, L., Fairlamb, A. H. and Cerami, A. (1990). Biochemistry 29, 3924.

Hewick, D. S . (1982). In “Metabolic Basis of Detoxication, Metabolism of Functional Groups’’ (W. B. Jacoby, J. R. Bend and J. Caldwell, eds), p. 151. Academic Press, New York.

Hoeoeg, J . O., Holmgren, A, , Silva, C. D., Douglas, K. T. and Seddon. A. P. (1982). FEBS Letters 138, 59.

Hoeoeg, J. O., Joernvall, H., Holmgren, A,, Carlquist, M. and Persson, M. (1983). European Journal of Biochemistry 136, 223.

Hoeoeg, J. O., von Bahr-Lindstroem, H. , Jocrnvall, H. and Holmgrcn, A. (1986). Genetics 43, 13.

Holmgren, A. (1976). Proceedings of the National Academy of Sciences of the United States of America 73, 2275.

Holmgren, A. (1979). Journal of Biological Chemistry 254, 3664. Holmgren, A. (1981). Trends in Biochemical Sciences 6, 26. Holmgren, A. (1983). In “Functions of Glutathione: Biochemical, Physiological,

Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 205. Raven Press, New York.

of Sciences of the United States of America 84, 439.

5, 210.

Clarendon Press, Oxford.

Biochemistry and Cell Biology 66, 288.

and Cell Biology 69, 115.

2182.

Holmgren, A. (1985). Annual Review of Biochemistry 54, 237. Holmgren, A. (1988). Biochemical Society Transactions 16, 95.

296 M. J . PENNINCKX AND M. T. ELSKENS

Holmgren, A,, Branden, C. I., Jornvall, H. and Sjoberg, B. M. (eds) (1986). “Thioredoxin

Hopkins, F. G. (1921). Biochemical Journal 15, 286. Hopper, D. J. and Cooper, R. A. (1971). FEBS Letters 13, 213. Iwami, K., Yasumoto, K. and Fushiki, T. (1978). Agricultural and Biological Chemistry 42,2175. Izuka, M., Murata, K. and Kimura, A. (1988). Agricultural and Biological Chemistry 52,613. Izuka, M., Inorie, Y., Murata, K. and Kimura, A. (1989). Journal of Bacteriology 171,6039. Jackson, P. J., Unkefer, C. J., Dovlen, J. A., Watt, K. and Robinson, N. J. (1987). Proceedings

Jacoby, W. B. (1978). Advances in Enzymology 36, 383. Jacoby, W. B. and Habig, W. H. (1980). In “Enzymic Basis of Detoxication” (W. B. Jacoby,

Jacoby, W. B., Ketterer, B. and Mannervik, B. (1984). Biochemical Pharmacology 33,2539. Jaspers, C. (1985). Ph.D. Thesis: Free University of Brussels. Jaspers, C. and Penninckx, M. (1981). FEBS Letters 132, 41. Jaspers, C. and Penninckx, M. (1982). Archives Internationales de Physiologie et de Biochimie

Jaspers, C. and Penninckx, M. (1984). Biochimie 66, 71. Jaspers, C., Gigot, D. and Penninckx, M. (1985). Phytochemistry 24, 703. Jocelyn, P. C. (1972). “Biochemistry of SH Group”. Academic Press, New York. Jockers-Scherubl, M. C., Schirmer, R . H . and Krauth-Siegel, R. L. (1989). European Journal

of Biochemistry 180, 267. Jones, E. W. and Fink, G . R. (1982). In “The Molecular Biology of the Yeast Saccharomyces

cerevisiae: Metabolism and Gene Expression” (J. N. Strathen, E. W. Jones and J. R. Broach, eds), pp. 181-299. Cold Spring Harbor Laboratory, New York.

and Glutaredoxin Systems: Structure and Function”. Raven Press, New York.

of the National Academy of Sciences of the United States of America 84, 6619.

J. R. Bend and J. Caldwell, eds), p. 63. Academic Press, New York.

90, 198.

Kaback, H. R. (1974). Science 186, 882. Kappus, H. (1985). In “Oxidative Stress’’ (H. Sies, ed.), p. 273. Academic Press, New York. Kappus, H. (1986). Biochemical Pharmacology 35 1. Kappus, H. (1987). Chemistry and Physics of Lipids 45, 105. Karplus, P. A. and Schulz, G. E. (1987). Journal of Molecular Biology 195, 701. Kato, H., Chihara, M., Nishioka, T., Murata, K., Kimura, K. and Oda, J . (1987). Journal

Kato, N., Sakazava, C., Nishizava, T., Tani, Y. and Yamada, H. (1980). Biochimica et

Kendall, E. C., Mackenzie, B. F. and Mason, H. L. (1929). Journal of Biological Chemistry

Kerklaan, P. R., Zoetemelk, C. E. andMohn, G. R. (1985). Biochemical Pharmacology 34,2151. Kistler, M., Summer, K. H. and Eckardt, F. (1986). Mutation Research 173, 117. Kistler, M., Maier, K. and Eckardt, F. (1990). Mutagenesis 5, 39. Kitamoto, K., Yoshizawa, K., Ohsumi, Y. and Anraku, Y. (1988). Journal of Bacteriology

Kosower, E. M. and Kanety-Londner, H. (1976). Journal of the American Chemical Society

Kosower, N. S. and Kosower, E. M. (1978). International Review of Cytology 54, 109. Kosower, E. M., Pazhenchevsky, B. and Hershkowitz, E. (1978). Journal of the American

Chemical Society 100, 6516. Kosower, N. S. , Kosower, E. M., Newton, G. L. and Ranney, H. M. (1979). Proceedings

of the National Academy of Sciences of the United States of America 76, 3382. Kosower, E. M., Kosower, N. S. and Radkwsky, A. (1983). In “Functions of Glutathione:

Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 243. Raven Press, New York.

Krauth-Siegel, R. L., Enders, B., Henderson, G. B., Fairlamb, A. H. and Schirmer, R. H. (1987). European Journal of Biochemistry 164, 123.

Kren, B., Parsell, D. and Fuchs, J. A. (1988). Journal of Bacteriology 170, 308. Kumagai, H . , Nakayama, R. and Tochikura, T. (1982). Agricultural and Biological Chemistry

of Biochemistry 101, 207.

Biophysica Acta 611, 323.

84, 657.

170, 2683.

98, 3001.

46, 1301.

GLUTATHIONE IN MICRO-ORGANISMS 297

Kumagai, H., Tamaki, H., Koshino, Y., Suzuki, H. and Tochikura, T. (1988). Agricultural

Kumar, K. S. , Sancho, A. M. and Weiss, J. F. (1986). International Journal of Radiation,

Kumar, S. , Ohja, V., Gangerly, N. K. and Kohti, K. K. (1990). Biochemistry International

Kunding, W. (1976). In “The Enzymes of Biological Membranes” (A. Martoni, ed.), p. 31.

Lahti, R. and Suonpaa, M. (1982). Journal of General Microbiology 128, 1023. Larsson, A, , Orrenius, S., Holmgren, A. and Mannervik, B. (eds) (1983). “Functions of

Glutathione: Biochemical, Physiological, Toxicologic.al, and Clinical Aspects”. Raven Press, New York.

and Biological Chemistry 52, 1377.

Oncology Biology, Physics 12, 1463.

20, 539.

Wiley, London.

Lau, E. P., Niswander, L., Watson, D. and Fall, R . (1980). Chemosphere 9, 565. Lawrence, R. A. and Burk, R. F. (1976). Biochemical and Biophysical Research

Lawrence, R. A. and Burk, R. F. (1978). Journal of Nutrition 108, 211. Levy, H. R. and Christoff, M. (1983). Biochemical Journal 214, 959. Li, L., Seddon, A. P. and Meister, A. (1988). Journal of Biological Chemistry 253, 1799. Llobell, A., Lopez-Ruiz, A, , Peinado, J. and Lopez-Barea, J. (1988). Biochemical Journal

Loewen, P. C. (1981). Canadian Journal of Biochemistry 59, 379. Lopez-Barea, J. and Lee, C. Y. (1979). European Journal of Biochemistry 98, 487. Mannervik, B. (1973). Biochemical and Biophysical Research Communications 53, 1151. Mannervik, B. (1982). In “Metabolic Basis of Detoxication” (W. B. Jakoby, J. R. Bend and

Mannervik, B. (1985). In “Advances in Enzymology and Related Areas of Molecular Biology”

Mannervik, B. and Axelsson, K. (1980). Biochemical Journal 190, 125. Mannervik, B. and Danielson, U. H. (1988). Critical Reviews in Biochemistry and Molecular

Biology 23, 283. Mannervik, B., Guthenberg, C., Jensson, H., Warholm, M. and A h , P. (1983). In “Functions

of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 75. Raven Press, New York.

Mark, D. F. and Richardson, C. C. (1976). Proceedings of the National Academy of Sciences of the United States of America 13, 780.

Mark, D. F., Chase, J. W. and Richardson, C. C. (1977). Molecular and General Genetics 155, 145.

Mason, R. P. and Josephy, P. D. (1985). In “Toxicity of Nitroaromatic Compounds” (D. E. Rickert, ed.), p. 21. Hemisphere, Washington, DC.

Masselot, M. and de Robichon-Szulmajster, H. (1975). Molecular and General Genetics 139, 121.

Masselot, M. and Surdin-Kerjan, Y. (1977). Molecular and General Genetics 154, 23. Mata, A. M., Pinto, M. C. and Lopez-Barea, J. (1985a). Molecular and Cellular Biochemistry

Mata, A. M., Pinto, M. C. and Lopez-Barea, J . (1985b). Molecularand Cellular Biochemistry

Mathews, C . K., Sjoberg, B. M. and Reichard, P. (1987). European Journal of Biochemistry

Mehra, R. K., Target, E. B., Gray, W. R. and Winge, D. R. (1988). Proceedings of the

Meister, A. (1974). In “The Enzymes” (P. D. Boyer, ed.), vol. 10, pp. 671-697. Academic

Meister, A. (1981a). Current Topics in Cellular Regulation 18, 21. Meister, A. (1981b). Trends in Biochemical Sciences 6, 231. Meister, A. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological,

Communications 71, 952.

249, 293.

J . Caldwell, eds), p. 185. Academic Press, New York.

(A. Meister, ed.), p. 357. Wiley, New York.

67, 65.

68, 121.

166, 279.

National Academy of Sciences of the United States of America 85, 8815.

Press, New York.

298 M. J. PENNINCKX AND M. T. ELSKENS

and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 1. Raven Press, New York.

Meister, A. (ed.) (1985). Methods in Enzymology 113. Meister, A. (1988). Journal of Biological Chemistry 263, 17205. Meister, A. and Anderson, M. E. (1983). Annual Review of Biochemistry 52, 711. Messenguy, F., Colin, D. and Ten Have, J . P. (1980). European Journal of Biochemistry

Meury, J. and Robin, A. (1985). European Journal of Biochemistry 143, 113. Meury, J. and Robin, A. (1990). Archives of Microbiology 154, 475. Meyer, M., Deitrichs, D., Schmidt, B. and Andreesen, J . R. (1991). Journal of Bacteriology

Milbauer, R. and Grossowicz, N. (1965). Journal of General Microbiology 41, 185. Minato, S. (1979). Archives of Biochemistry and Biophysics 192, 235. Mitchell, J. B. (1988). IS1 Atlas of Science: Pharmacology, 155. Mohn, G., de Kniff, P. and Baars, A. (1983). Mutation Research 111, 25. Moncada, C., Repetto, Y . , Aldunate, J., Letelier, M. E. and Morello, A. (1989). Comparative

Monks, T. J., Anders, M. W., Dekant, W., Stevens, J. L., Lau, S. and van Bladeren, P. J.

Montero, S. , de Arriaga, D. and Soler, J. (1988). Biochimica et Biophysica Acta 952, 56. Montero, S . , de Arriaga, D., Busto, F. and Soler, J. (1990). Archives of Biochemistry and

Moore, W. R., Anderson, M. E., Meister, A, , Murata, K. andKimura, A. (1989). Proceedings

Mooz, E. D. (1979). Biochemical and Biophysical Research Communications 90, 1221. Mooz, E. D. and Meister, A. (1967). Biochemistry 6, 1722. Mooz, E. D. and Wigglesworth, L. (1976). Biochemical and Biophysical Research

Morse, M. L. and Dahl, R. H. (1978). Nature 271, 654. Morzycka, E. and Paszewski, A. (1982). Acta Biochimica Polonica 29, 81. Murata, K . and Kimura, A. (1990). Biotechnology Advances 8, 59. Murata, K., Tani, K., Kato, J. and Chibata, I. (1980). Journal of General Microbiology 120,

Murata, K., Tani, K., Kato, J . and Chibata, I. (1981). Agricultural and Biological Chemistry

Murata, K., Inorie, Y., Watanabe, K., Fukuda, Y . , Saikusa, T. and Kimura, A. (1986a).

Murata, K., Saikusa, T., Watanabe, K., Fukuda, Y., Shimosaka, M., Inorie, Y. and Kimura,

Murata, K., Sato, N., Rhee, H. I., Watanabe, K. and Kimura, A. (1987). Agricultural and

Murray, H. W., Aley, S. B. and Scott, W. A. (1981). Molecular and Biochemical Parasitology

Nagai, S. and Black, S. (1968). Journal of Biological Chemistry 243, 1942. Nakayama, R. (1984). Ph.D. Thesis: Kyoto Univcrsity. Nakayama, R., Kumagai, H. and To Chikura, T. (1984). Applied and Environmental

Neben, I., Sahm, H. and Kula, M. R. (1980). Biochimica et Biophysica Acta 614, 81. Newton, G. L. and Fahey, R. C. (1990). In “Glutathione: Metabolism and Physiological

Newton, G. L. and Javor, B. (1985). Journal of Bacteriology 161, 438. Nikolaev, 0. N., Alver’Yanov, A. A. and Dzhavakhiya, V. G. (1989). Mikologija i

Noda, K., Igato, K., Horikawa, Y. and Fijii, M. (1980). Agriculturaland Biological Chemistry

Ogawa, T., Oka, Y. and Sasaoka, K. (1987). Journal of Food Science 52, 135.

108, 439.

173, 1509.

Biochemistry and Physiology 94, 87.

(1990). Toxicology and Applied Pharmacology 106, 1.

Biophysics 278, 52.

of the National Academy of Sciences of the United States of America 86, 1461.

Communications 68, 1066.

545.

45, 2131.

Agricultural and Biological Chemistry 50, 135.

A. (1986b). European Journal of Biochemistry 57, 297.

Biological Chemistry 51, 1901.

3, 381.

Microbiology 47, 653.

Functions” (J. Vina, ed.), p. 69. CRC Press, Boca Raton.

Fitopatologija 23, 439.

44, 2419.

GLUTATHIONE IN MICKO-ORGANISMS 299

Ogawa, T., Oka, Y. and Sasaoka, K. (1990). Phytochemistry 29, 303. Ohtake, Y. and Yabuuchi, S. (1991). Yeast 7, 953. Ohtake, Y . , Watanabe, K., Tezuka, H., Ogata, T., Yabuuchi, S . , Murata, K. and Kimura,

Ondarza, R . N., Rendon, J. L. and Ondarza, M. (1983). Journal of Molecular Evolution 19,

Ono, B., Naito, K., Shirahige, Y. and Yamamoto, M. (1991). Yeast 7, 843. Ono, B. I., Shirage, Y. I . , Nanjoh, A, , Andou, N., Ohue, H. and Ishino-Arao, Y. (1988).

Orlowski, M. and Meister, A. (1971). Journal of Biological Chemistry 246, 7095. Osuji, G. 0. (1980). FEBS Letters 110, 192. Overbaugh, J. M. (1985). Journal of Free Radicals and Biological Medicine 1, 187. Overbaugh, J. M. and Fall, R. (1982). FEMS Microbiology Letters 13, 371. Overbaugh, J. M. and Fall, R. (1985). Plant Physiology 77, 437. Overbaugh, J. M., Lau, E. P., Marino, V. A. and Fall, R. (1988). Archives of Biochemistry

Pai, E. F. and Schulz, G. E. (1983). Journal of Biological Chem&try 258, 1752. Paszewski, A. and Grabski, J. (1974). Molecular and General Genetics 132, 307. Paszewski, A. and Grabski, J . (1975). Journal of Bacteriology 124, 893. Payne, G. M. and Payne, J. W. (1984). Biochemical Journal 218, 147. Pel, R. and Gottschal, J. C. (1989). FEMS Microbiology Ecology 62, 349. Penketh, P. G. and Klein, R. A. (1986). Molecular and Biochemical Parasitology 20, 111. Penketh, P. G., Kennedy, W. K., Patton, C. L. and Sortorelli, A. (1987). FEBS Letters 221,

Penninckx, M. J. and Jaspers, C. J. (1982). Bulletin de l‘fnstitut Pasteur (Paris) 80, 291. Penninckx, M. J. and Jaspers, C. J. (1985). Phytochernistry 24, 1913. Penninckx, M. J., Jaspers, C. J. and Wiame, J . M. (1980). European Journal of Biochemistry

A. (1989). Journal of Fermentation and Bioengineering 68, 390.

371.

Journal of Bacteriology 170, 5883.

and Biophysics 261, 227.

427.

104. 119. , ~~

Penninckx, M. J., Jaspcrs, C. J . and Lcgrain, M. (1983). Journal of Biological Chemistry 258, 6030.

Perham, R. N., Berry, A. and Scrutton, N. S . (1988). Biochemical Society Transactions 16, 84.

Perry, A. C., Bhriain, N. N., Brown, N. L. and Rouch, D. A. (1991). Molecular Microbiology 5 , 163.

Piccolomini, R., Di Ilio, C., Aceto, A, , Allocati, N., Faraone, A,, Cellini, L., Ravagnan, G. and Federici, G. (1989). Journal of General Microbiology 135, 3119.

Pickett, C. B. and Lu, A. Y. (1989). Annual Review of Biochemistry 58, 743. Pinto, M. C., Mata, A. M. and Lopez-Barea, J. (1984). Archives of Biochemistry and

Pinto, M. C., Mata, A. M. and Lopez-Barea, J. (1985). European Journal of Biochemistry

Price, N. W. and Pinhey, K. G. (1929). Journal of Biological Chemistry 84, 321. Prohaska, J. R. (1980). Biochimicu ef Biophysica Acra 611, 87. Pryor, W. A. (1962). “Mechanisms of Sulfur Reactions”. McGraw-Hill, New York. Racker, E. (1951). Journal of Biological Chemistry 190, 685. Ramaswamy, O., Pal, S. , Guha-Mukerjee, S . and Sojory, S . K. (1983). Biochemistry

Reed, D. J. (1986). Biochemical Pharmacology 35, 7. Reed, D. J., Brodie, A. E. and Meredith, M. J. (1983). In “Functions of Glutathione:

Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannevik, eds), p. 39. Raven Press, New York.

Biophysics 228, 1.

151, 275.

International 7, 307.

Reichard, P. (1987). Biochemistry 26, 3245. Reichard, P. and Thelander, L. (1979). Annual Review of Biochemistry 48, 133. Rennenberg, H. (1982). Phytochemistry 21, 2771. Repetto, Y., Letelier, M. E., Aldunate, J. and Morello, A. (1987). Comparative Biochemistry

and Physiology 87, 73.

300 M. J. PENNINCKX AND M. ‘r. ELSKENS

Revesz, L. and Malaise, E. P. (1983). In “Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 163. Raven Press, New York.

Rhee, H. I., Murata, K. and Kimura, A. (1986). Biochemical and Biophysical Research Communications 141, 993.

Rhee, H. I . , Sato, N., Murata, K. and Kimura, A. (1988). Agricultural and Biological Chemistry 52, 2243.

Richman, P. and Meister, A. (1975). Journal of Biological Chemistry 250, 1422. Rizza, V . and Hu, A. S . L. (1973). Biochemical and Biophysical Research Communications

54, 168. Roberts, R. B., Abelson, P. H., Cowie, D . B., Bolton, E. T. and Britten, R. J. (1963).

“Studies of Biosynthesis in Escherichia coli”, p. 424. Carnegie Institute, Washington, DC. Robins, R. and Davies, D . (1981a). Biochemical Journal 194, 63. Robins, R. and Davies, D. (1981b). Phytochemistry 20, 1497. Ropenga, J. S. and Lenfant, M. (1987). Applied Microbiology and Biotechnology 26, 117. Ropenga, J. S. , Yax, P. and Lenfant, M. (1989). Applied Microbiology and Biotechnology

Rose, Z . B. and Racker, E . (1962). Journal of Biological Chemistry 237, 3279. Roseman, S . (1969). Journal of General Microbiology 54, 138. Russel, M. and Holmgren, A. (1988). Proceedings of the National Academy of Sciences of

Sahm, H. (1977). Advances in Biochemical Engineering 6 , 77. Sandberg, V . A,, Kren, B., Fuchs, J. A. and Woodward, C. (1991). Biochemistry 30, 5475. Saxena, M., Mukerji, K. G. and Raj, H. G. (1988). Biochemical Journal 254, 567. Scherens, B. , Dubois, E. and Messenguy, F. (1991). Yeast 7 , 185. Schwartz, R., Lucas, M. T., Escalante, L., Vasquez, G. and Sanchez, S . (1988). Journal of

Schutte, H., Flossdorf, J., Sahm, H. and Kula, M. R. (1976). European Journal of

Scrutton, N. S. , Berry, A. and Perham, R. N. (1987). Biochemical.iournal245, 875. Sels, A. A. and Brygier, J. (1980). European Journal of Biochemistry 112, 283. Serrano, A,, Rivas, J. and Losada, M. (1984). Journal of Bacteriology 158, 317. Shames, S . L., Fairlamb, A. H., Cerami, A. and Walsh, C. T. (1986). Biochemistry 25,3519. Shames, S . L., Kimmel, B. E., Peoples, 0. P., Agabian, N. and Walsh, C. T. (1988).

Shimuzi, H., Araki, K., Shioya, S. and Suga, K. (1991). Biotechnology and Bioengineering

Shishido, T. (1981). Agricultural and Biological Chemistry 45, 2951. Smith, C . V., Hughes, H., Lauterburg, B. H. and Mitchell, J . R. (1983). In “Functions of

Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S . Orrenius, A. Holmgren and B. Mannervik, eds), p. 125, Raven Press, New York.

31, 176.

the United States of America 85, 990.

General Microbiology 134, 1117.

Biochemistry 62, 151.

Biochemistry 27, 5014.

38, 196.

Smith, J. and Shrift, A. (1978). Comparative Biochemistry and Physiology 63, 39. Soda, K. (1987). Methods in Enzymology 143, 453. Spallholz, J. E. (1987). Medical Hypotheses 23, 253. Stadtman, E. R. and Kornberg, A. (1953). Journal of Biological Chemistry 203, 47. Summer, K. H., Goeggelmann, W. and Greim, H. (1980). Mutation Research 70, 269. Sundquist, R. A. and Fahey, R. C. (1988). Journal of Bacteriology 170, 3459. Sundquist, R. A. and Fahey, R . C. (1989). Journal of Molecular Evolution 29, 429. Suzuki, H., Kumagai, H. and Tochikura, T. (1986). Journal of Bacteriology 168, 1325. Suzuki, H., Kumagai, H., Echigo, T. and Tochikura, T. (1989). Journal of Bacteriology 171,

Swerdlow, R. D. and Setlow, P. (1983). Journal of Bacteriology 153, 475. Sysoev, 0. V., Govorukhina, N. I. and Gruzman, M. (1990). Prikladnaia Biokhimiia I

Szajewski, R. P. and Whitesides, G. M. (1980). Journal of American Chemical Society 102,

5169.

Mikrobiologiya 26, 456.

2011.

GLUTATHIONE IN MICKO-ORGANISMS 301

Tabor, H. and Tabor, C. W. (1975). Journal of Biological Chemistry 250, 2648. Tabor, H. and Tabor, C. W. (1979). Biochemical and Biophysical Research Communications

Tamaki, H. , Kumagai, H. and Tochikura, T. (1989). Journal of Bacteriology 171, 1173. Tate, S. S. and Meister, A. (1978). Proceedings of the National Academy of Sciences of the

Taylor, M. C., Chapman, C. J., Kelly, J. M., Fairlamb, A. H. and Miles, M. A. (1989).

Tel-Or, E. , Huflejt, M. and Packer, L. (1985). Biochemical and Biophysical Research

Thornalley, P. J. (1990). Biochemical Journal 269, 1. Tomita, K., Ito, M., Yano, T., Kumagai, H. and Tochikura, T. (1988). Agricultural and

Tomita, K., Yano, T., Kitagata, T . , Kumagai, H. and Tochikura, T. (1989). Agriculturaland

Tomizawa, H. H. (1962). Journal of Biological Chemistry 237, 3393. Tsai, C. S. and Godin, G. R. (1987). International Journal of Biochemistry 19, 337. Uotila, L. and Koivusalo, M. (1974). Journal of Biological Chemistry 249, 7653. Uotila, L. and Koivusalo, M. (1983). In “Functions of Glutathione: Biochemical,

Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 175. Raven Press, New York.

41, 232.

United States of America 72, 1985.

Biochemical Society Transactions 17, 579.

Communications 132, 533.

Biological Chemistry 52, 11 59.

Biological Chemistry 53, 1995.

van Bladeren, P. J. and van Omen, B. (1991). Pharmacology and Therapeutics 51, 35. van Dijken, J . P., Oostra-Demkes, G. J., Otto, R. and Harder, W. (1976). Archives of

van Dijken, J . P., Harder, W. and Quayle, J. R. (1981). In “Microbial Growth on C1

Van Huffel, C. and Sels, A. A. (1987). Archives Internationales de Physiologie etde Biochimie

Verduyn, C., Giuseppin, M. L. F., Scheffers, W. A . andvanDijken, J. P. (1988). Appliedand

Von Sonntag, C. (1987). “The Chemical Basis of Radiation Biology”. Taylor and Francis,

Vos, R. M. and van Bladeren, P. J. (1990). Chemical and Biological Interactions 75, 241. Wackett, L. P. and Gibson, P. T. (1982). Biochemical Journal 205, 117. Watanabe, K., Murata, K. and Kimura, A. (1986). Agriculturaland Biological Chemistry 50,

Waxman, D. J. (1990). Cancer Research 50, 6449. Wendel, A. (1980). In “Enzymic Basis of Detoxication” (W. B. Jacoby, J. R. Bend and J.

Wiame, J . M., Grenson,M. andArst,H. N., Jr (1985).Advancesin Microbial Physiology26,2. Wiebers, J. L. and Garner, H. R. (1967). Journal of Biological Chemistry 242, 12. Wiemken, A. (1980). In “Cell Compartmentation and Metabolic Channeling”, p. 225.

Williams, C. H. (1976). In “The Enzymes” (P. D. Boyer, ed.), vol. 13, pp. 89-132. Academic

Williams, C. H. and Arscott, L. D . (1971). Methods in Enzymology 17B, 503. Yamada, Y., Tani, K. and Kamikara, T. (1984). Journal of General Microbiology 130,3275. Yamagata, S. (1980). Journal of Biochemistry 88, 1419. Yan, N. and Meister, A. (1990). Journal of Biological Chemistry 265, 1588. Yawetz, Z . A. and Agosin, M. (1981). Comparative Biochemistry and Physiology 68, 237. Yokota, A., Shigeoka, S. , Onishi, T. and Kitaoka, S. (1988). Plant Physiology 86, 649. Zhang, Y., Hempelmann, E. and Schirmer, R . H. (1987). Biochemical Pharmacology 37,855. Ziegler, D. M., Poulsen, L. L. and York, B. M. (1983). In “Functions of Glutathione:

Biochemical, Physiological, Toxicological, and Clinical Aspects” (A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik, eds), p. 205. Raven Press, New York.

Microbiology 111, 77.

Compounds” (H. Dalton, ed.), pp. 199-201, Heyden, London.

95, 96.

Environmental Microbiology 54, 2086.

London.

1925.

Caldwell, eds), p. 333. Academic Press, New York.

ElsevierINorth Holland Biomedical Press, Amsterdam.

Press, New York.