APPROVED: Daniel A. Kunz, Major Professor Barney J. Venables, Committee Member Gerard A. O’Donovan, Committee Member Arthur J. Goven, Chair of the Department of

Biological Sciences Sandra L. Terrell, Interim Dean of the Robert B.

Toulouse School of Graduate Studies

BACTERIAL CYANIDE ASSIMILATION: PTERIN COFACTOR AND ENZYMATIC

REQUIREMENTS FOR SUBSTRATE OXIDATION

Elena Dolghih, B.S.

Thesis Prepared for the Degree of

MASTER OF SCIENCE

UNIVERSITY OF NORTH TEXAS

May 2004

Dolghih, Elena, Bacterial Cyanide Assimilation: Pterin Cofactor and

Enzymatic Requirements for Substrate Oxidation. Master of Science (Molecular

Biology), May 2004, 54 pp., 3 tables, 11 figures, 1 scheme, references, 41 titles.

Utilization of cyanide as the sole nitrogen source by Pseudomonas

fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon

dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate

attack is initiated oxygenolytically by an enzyme referred to as cyanide

oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase.

The pterin requirement for Pf11764 CNO was satisfied by supplying either the

fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds

at catalytic concentrations (0.5 µM). These compounds included, for example,

biopterin, monapterin and neopterin, all of which were also identified in cell

extracts. A related CNO-mediated mechanism of cyanide utilization was

identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i)

the recovery of CO2 and NH

3 as enzymatic reaction products, (ii) the dependency

of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O

2

and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for

Pf11764, it was not possible to demonstrate a need for exogenously added pterin

as a cofactor for the PpBCN3 enzyme system. However, results which showed

that cells of PpBCN3 contained approximately seven times the amount of pterin

as Pf11764 (of which a significant portion was protein-bound) were interpreted as

indicating that sufficient bound CNO-cofactor exists, thus eliminating any need

for a supplemental source.

ii

ACKNOWLEDGMENTS

I would like to express my gratitude to Dr. Kunz, who guided me in my

research, and provided encouragement and inspiration. I would also like to thank

my parents and my sister for moral support and for believing in me. And lastly, I

wish to express my gratitude to Ruby, my fellow graduate student, for all her help.

iii

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS......................................................................................ii LIST OF TABLES ................................................................................................. v LIST OF FIGURES...............................................................................................vi LIST OF SCHEMES ............................................................................................vii Chapter I. INTRODUCTION............................................................................. 1

Cyanide in Nature................................................................. 1

Cyanide Tolerance ............................................................... 1

Enzymology of Cyanide Assimilation in P. fluorescens NCIMB 11764....................................................................... 3

II. MATERIALS AND METHODS ........................................................ 9

Bacterial Strains ................................................................... 9

Cultivation Conditions........................................................... 9

Preparation and Fractionation of Cell Extracts ..................... 9

Cyanide Depletion and Product Formation......................... 10

Time-dependent Assay of CNO.......................................... 13

Analytical Methods ............................................................. 14

Pterin Analysis.................................................................... 17

Materials and Supplies ....................................................... 20 III. RESULTS Part 1: CNO from P. fluorescens NCIMB 11764 Shows a

Relaxed Specificity for Pterin Cofactors ............................. 22

Pf11764 produce pterins of the biopterin, monapterin and neopterin series ................................................ 22

Cofactor activity of pterin compounds in Pf11764.... 31

iv

Part II: Cyanide Oxidation in P. putida BCN3 Involves a CNO-Mediated Enzymatic Mechanism............................... 33

Recovery of cyanide oxidation activity in PpBCN3 cell-free extracts ...................................................... 33

Cyanide oxidation by fractionated P. putida BCN3 cell extracts .................................................................... 36

P. putida BCN3 synthesizes pterins of the biopterin, monapterin and neopterin series at levels approximately seven times that of Pf11764 ............. 43

IV. DISCUSSION................................................................................ 47 REFERENCES................................................................................................... 51

v

LIST OF TABLES

Page

1. CNO-enhancing activities of pterin compounds in P. fluorescens............ 32

2. Products of cyanide metabolism by cell-free extracts of P. putida ........... 40

3. Pterin content of cyanide degrading bacteria........................................... 46

vi

LIST OF FIGURES

Page

1. General structures of fully reduced, partially reduced, and fully oxidized pterins........................................................................................................ 7

2. Fractionation of cell extracts of Pf11764 into high (H) and low (L) molecular weight components required for cyanide oxidation activity...... 11

3. Standard curve for ammonia determination ............................................. 15

4. Standard curve for determination of oxidized pterins by fluorescence high pressure liquid chromatography............................................................... 18

5. HPLC analysis of the filtrate (10 ml) of P. fluorescens obtained after passage of P. fluorescens HSS through a 10 kDa cutoff ultraconcentrator...................................................................................... 23

6. UV spectra of biologically produced H2biopterin and commercial standards H4biopterin and H2biopterin.................................................... 26

7. Effect of biopterins on cyanide oxidation activity by cell extracts of P. fluorescens .......................................................................................... 29

8. Time course of cell-free cyanide consumption by P. putida ..................... 34

9. Cyanide and formate conversion to CO2 by cell extracts of P. putida ..... 37

10. Cyanide oxidation activity measured as NADH oxidation and O2 consumption ............................................................................................ 41

11. High pressure liquid chromatography analysis of oxidized HSS of P. putida ....................................................................................................... 44

vii

LIST OF SCHEMES

Page

1. Proposed pathway for cyanide oxidation in P. fluorescens........................ 4

1

CHAPTER I

INTRODUCTION

Cyanide in Nature

Cyanide is a toxic compound that binds to cytochrome oxidases inhibiting

electron transport (Knowles, 1988). It can also disrupt the functions of a variety of

other enzymes due to its high reactivity towards important functional groups and

metal centers (Solomonson, 1981). Notwithstanding such toxic properties,

cyanide is rather common in the environment. It is produced in the form of

cyanogenic glycosides by plants such as maize, millet, sorghum and sweet

potatoes and a number of fungi and bacteria are also known to be cyanogenic

(Knowles and Bunch, 1986; Castric, 1981; Poulton, 1988). In addition, the use

of cyanide in various industrial operations such as metal plating and gold mining

represent anthropogenic sources (Homan, 1987).

Cyanide Tolerance

While most organisms are very sensitive to cyanide, others not only

tolerate it but, in some cases, even use it as a source of nutrition. Cyanide

tolerance can be imparted by two means: 1) by the synthesis of cyanide resistant

cytochrome oxidases (Bonner et al., 1986; Cunningham and Williams, 1995) and

2) by breakdown of cyanide to nontoxic products. From mammals to

microorganisms, there exists a variety of enzymes capable of detoxifying cyanide.

2

rhodanase

The enzyme rhodanase (E C 2.8.1.1), for example, catalyzes the conversion of

cyanide to less toxic thiocyanate by addition to thiosulfate. Some organisms

S2O3- + HCN HSCN + SO3

-2 eq. 1

produce β-cyanoalanine synthase (E C 4.2.99.8) which catalyzes the

nucleophilic substitution of a thiol in cysteine for cyanide (eq. 2) (Castric, 1981).

HCN + cysteine β-cyanoalanine + acetate eq. 2

The enzyme cyanide hydratase (E C 4.2.1.66), found extensively among

phytopathogenic fungi, adds water across the cyanide triple bond to give

formamide (eq. 3) (Fry and Millar, 1972; Wang et al., 1992). Two enzymes are

HCN + H2O HCONH2 eq. 3

also known that break down cyanide to ammonia, a universal nitrogen source

that can then potentially be used for growth. One such enzyme is cyanide

dihydratase (CynD) (also known as cyanide nitrilase or cyanidase) whose second

product is formate. CynD has been reported in Alcaligenes xylosoxidans

(Ingvorsen et al., 1991), Bacillus pumilus (Meyers et al., 1993) and

Pseudomonas stutzeri (Jandhyala et al., 2003). Because this enzyme functions

predominantly at high substrate concentrations that are likely to be lethal (Km =

1.5-10 mM) (Raybuck, 1992), it is unclear whether CynD imparts any ability for

bacteria to grow on cyanide. A second mechanism of cyanide conversion to

cyanide hydratase

β-cyanoalanine synthase

3

HCN + 2H2O HCO2H + NH3 eq. 4

ammonia is that involving its oxidative conversion to CO2 and NH3. This pathway

has been described in Pseudomonas fluorescens (NCIMB 11764) and shown to

be initiated oxygenatively by an enzyme referred to as cyanide oxygenase (CNO)

(Harris and Knowles, 1983; Knowles, 1988; Kunz et al., 1994; Kunz et al., 2001).

HCN + O2 + NADH +H+ CO2 + NH3 + NAD+ eq. 5

The properties of CNO and biochemistry of cyanide assimilation in Pf11764

represent active areas of investigation in our laboratory and are further reviewed

below.

Enzymology of Cyanide Assimilation in P. fluorescens NCIMB 11764

Pseudomonas fluorescens NCIMB 11764 (Pf11764) was first reported to

be able to grow on cyanide by Knowles and Harris in 1983. The cell-free

conversion of cyanide to ammonia and CO2 was shown to require oxygen and

reduced pyridine nucleotide (NADH), which was interpreted as indicating that the

responsible enzyme was an oxygenase (CNO). Later studies by Kunz et al.

(1994) with mutant strains correlated the lack of growth on cyanide with an

inability to produce CNO activity, thus indicating that CNO is absolutely required

for growth. To prove that CNO functions as a true oxygenase, isotopically-

labelled 18O2 studies by Wang, Kunz and Venables (1996) established that only

cyanide dihydratase

cyanide oxygenase

4

one of the atoms in the reaction product, CO2, was derived from molecular

oxygen. Related studies showing that the second oxygen atom originated from

water, suggested that cyanide conversion occurs by two-step reaction

mechanism involving the formation of a monooxygenated intermediate whose

subsequent hydrolysis gives rise to NH3 and CO2 (eqs. 6 and 7). Separate

HCN + O2 + NADH + H+ [X-OH] + H2O + NAD+ eq. 6

[X-OH] + H2O CO2 + NH3 eq. 7

studies showed that a second enzyme, formate dehydrogenase (FDH), was also

involved since FDH was co-induced with CNO and catalyzed the oxidation of

formate to CO2. Recent efforts by R. Fernandez (Fernandez, 2004) resulting in

the resolution of CNO and FDH enzymes in Pf11764, have further confirmed a

reaction pathway in which cyanide is oxidized to CO2 via formate. The overall

reaction stoichiometry in which one molar equivalent each of O2 and NADH is

utilized in complete substrate oxidation is shown in Scheme 1.

HCN

2NAD+

HCO2H CO2

NAD+2NADH + H+ NADH + H+

O2 NH3

CNO FDH

During studies aimed at characterizing the CNO enzyme, it was

discovered that enzyme activity was dependent on a small mol wt cofactor for

Scheme 1

5

which 5,6,7,8-tetrahydro-L-biopterin (H4biopterin) was able to substitute (Kunz et

al., 2001). Pterins, being a group of GTP-derived compounds (see Fig. 1), are

common in biology and have been described in one form or another in a variety

of organisms (Nixon, 1985). Their biological function is primarily one of serving

as cofactors for enzymes of which the amino acid hydroxylases (AAH) (E C

1.14.16.1) (Kappock and Caradonna, 1996) and nitric oxide synthase (NOS) (E C

1.14.13.39) (Marletta et al., 1998) are most notable. Both AAHs and NOS rely on

tetrahydrobiopterin as a cofactor in catalyzing monooxygenation reactions.

Hence, the requirement by CNO for H4biopterin was interpreted as indicating that

the enzyme functions as a pterin-dependent hydroxylase (Kunz et al., 2001).

Studies aimed at identifying the natural cofactor for the enzyme resulted in the

identification of H4biopterin in cell extracts (Fernandez, 2004). However,

additional pterin species of unknown identity were also detected raising the

possibility that these compounds might also serve as cofactors for the enzyme.

This study describes the identification of these compounds and shows that the

Pf11764 CNO has a relaxed specificity towards pterin cofactors. Because

Pf11764 is thus far the only organism in which cyanide assimilation has been

shown to proceed by a CNO-mediated oxidative mechanism, separate studies on

an additional cyanide-degrading bacterium, P. putida BCN3, were performed in

order to determine the enzymatic basis of cyanide assimilation in this organism.

P. putida BCN3, like Pf11764 was found also to metabolize cyanide by a putative

CNO-catalyzed process. However, it was not possible to demonstrate

6

conclusively, that in this case cyanide oxidation is also pterin-dependent because

of difficulties associated with the removal of pterin compounds from extracts due

to their high concentration. Besides the description of a second CNO enzyme

system in bacteria, the identification of pterins compounds previously

unrecognized in Pseudomonas bacteria is further reported.

7

Figure 1. General structures of fully reduced, partially reduced and fully oxidized

pterins. Monapterin (6-L-threo-(1,2,3-trihydroxypropyl)pterin) series, neopterin (6-

L-erythro)-(1,2,3-trihydroxypropyl)pterin) series, and biopterin (6-L-erythro-(1,2-

dihydroxypropyl)pterin) series.

8

HN

H N

O

N H

NH2N 12

35

6

78

4R

H

HH

Pterin

H4pterin H2pterin

R = R =

Monapterin series Biopterin series

HNN

O

NNH2N 12

35

6

78

4R

C C CH3

H

OH

H

OH

C C CH2OH

OH

H

H

OH

HNN

O

N H

NH2N1

2

35

6

78

4R

HH

C C CH2OH

OH

H

OH

H

Neopterin series

R =

9

CHAPTER II MATERIALS AND METHODS

Bacterial Strains

P. fluorescens NCIMB 11764 (Pf11764) was originally acquired (1989)

from the National Collection of Marine and Industrial Bacteria, Torrey, Scotland.

Pseudomonas putida BCN3 (PpBCN3) was isolated in our laboratory by J. Silva-

Avalos (Silva-Avalos et al., 1990). All strains were stored at -80°C.

Cultivation Conditions

Frozen stock cultures of Pf11764 were stored at -80°C and subcultured

onto Lennox agar plates (Lennox, 1955). After 36 hours of incubation at 30°C, a

single colony was picked and inoculated to minimal medium containing 10 mM

glucose as a carbon source and 1 mM ammonium chloride as a nitrogen source.

The culture was incubated for 48 hours on a shaker at 200 rpm at 30°C and

transferred (10% inoculum) to a flask with the same medium, which, after 24-

hour incubation, was induced for CNO by the addition of 0.1 mM KCN. After 3

hour, cells were harvested and washed in 50 mM Na2HPO4-KH2PO4 (Na-K) (pH

7.0) buffer before being stored at -80°C until use.

Preparation and Fractionation of Cell Extracts

Frozen cells were resuspended in twice the volume of Na-K buffer and

10

broken in a French Press at 20,000 psi. After incubating the mixture for 5 minutes

at room temperature with a small amount of DNAse to remove nucleic acids,

extracts were subjected to centrifugation at 30,000 x g for 30 minutes and the

supernatants subsequently centrifuged at 150,000 x g for 90 minutes. Extracts

were further fractionated as shown in Fig. 2 by membrane ultrafiltration

(Centricons, Amicon, Danvers, MA). Following passage through a Centricon-30

at 4˚C for 1 hour at 1500 x g , the retentate (Fraction H) was retained and the

filtrate passed further through a Centricon-10. The resultant filtrate (Fraction L)

was collected and both it and fraction H were stored until use at -80°C.

To further clarify PpBCN3 extracts, HSS proteins were suspended in Na-K

buffer containing an anion-exchange support (Sepharose) (1/5 the volume), and

after removal of non-bound material (lipid + non-binding proteins), enzyme

activity was removed from the support by elution with 0.5 M Na2SO4. The protein

was desalted with Na-K buffer using Centricon-10 (<10 kDa cutoff) ultrafiltration

device.

Cyanide Depletion and Product Formation

Cyanide conversion to CO2 was tested by incubating cell extracts with

K14CN in sealed containers on a gyrorotary shaker. Reaction mixtures (0.25-1

mg protein) were incubated in Na-K buffer (0.05-0.25 ml) in a 2-ml HPLC vial

(Varian, Walnut Creek, CA), which was placed inside a 15-ml crimp-seal bottle

11

Figure 2. Fractionation of cell extracts of Pf11764 into high (H) and low (L)

molecular weight components required for cyanide oxidation activity.

12

13

(Pierce, Rockford, IL) that was sealed. NADH (0.2-1 mM) was added to the

reaction contents by syringe followed by the injection of 0.1 mM K14CN (1 µCi,

sp.act., 56 mCi mmol-1) to initiate the reaction. Reactions were terminated by the

injection of 6M ZnSO4 (5 �l) into the inner HPLC vial, and immediately thereafter,

0.4 ml of 4N NaOH was injected into the outer crimp-seal bottle compartment to

collect volatile 14CO2. Following a 30 minute incubation period, the bottle was

opened and the contents of both the inner HPLC vial and crimp-seal bottle were

fractionated with BaCl2 and radioactivity recovered as previously described (Kunz

et al., 1992). The % 14CO2 yield was calculated as the ratio of counts collected as

volatile 14CO2 (outer compartment barium precipitable fraction) over the total.

Time-dependent Assay of CNO

The standard assay for CNO activity was measurement of cyanide

consumption in sealed vials incubated on a shaker at 30oC. Reaction mixtures

(0.12-0.25 ml) contained 2-4 mg per ml protein, 0.2-1 mM NADH and 0.1 mM

KCN in total volume of 0.12-0.25 ml of Na-K buffer. Reactions were initiated by

the injection of KCN and at timed intervals samples were withdrawn and the

remaining cyanide determined. CNO activity was also assayed by measuring

the cyanide-dependent consumption of NADH by O2. NADH consumption was

measured spectrophotometrically, where reaction mixtures contained 0.2 mM

NADH and 0.5-1mg of protein in 0.5 ml of Na-K buffer. Upon addition of KCN (10

nmol), NADH oxidation was monitored at A340 at ambient temperature. Oxygen

14

consumption was measured at 30°C using Clark-type O2-electrode (Rank Bros.,

UK). Standard reaction mixtures contained the same components as above in

air-saturated buffer (0.224 mM O2).

Analytical Methods

Ammonia

Incubations were terminated by addition of 10 µL of 6M ZnSO4 and after

removal of the protein in a microfuge, ammonia was determined according to the

Fawcett and Scott method (Fawcett and Scott, 1960). To 125 µl of sample was

added 0.25 ml Na phenate (2.5% Phenol and 7.8% 4N NaOH in water), 0.375 ml

of 0.01% Na nitroprusside and 0.375 ml of 0.2 N NaOCl. After 30 minutes, the

A630 was determined and ammonia quantified from a standard curve (Fig. 3).

Cyanide

Cyanide was determined by the Lambert method (Lambert et al., 1975).

Samples (50 µl) were added to 1.1 ml of water containing 50 µl of N-

chlorosuccinimide/succinimide (0.1 and 1% w/v, respectively). To this mixture, 50

µl of barbituric acid reagent (6% in 30% aqueous pyridine) was added and the

A580 determined after 15 minutes.

Proteins

The protein content of extracts was determined using the Lowry method

(Lowry et al., 1951). The standard procedure involved adding 5 µl of protein

15

Figure 3. Standard curve for ammonia determination (y = 0.002x – 0.005).

16

0

0.1

0.2

0.3

0.4

0.5A

bsor

banc

e(6

30 n

m)

0 50 100 150 200 250NH4Cl(µΜ))))

17

sample to 0.2 ml of water to which was added reagent A (prepared fresh by

mixing 0.5% CuSO4·5H2O made in 1% sodium tartrate with 2% Na2CO3 prepared

in 0.1 N NaOH in 1:50 ratio). After 10 minutes, 0.1 ml of 1N Folin-phenol reagent

(2N) was added and the absorbance read at 750 nm after 30 minutes. The

sample protein concentration was determined using a standard curve obtained

with bovine serum albumin as a standard.

Pterin Analysis

High pressure liquid chromatography

Samples (10-20 �l) were injected onto a Microsorb C18 column (4.6 x 250

mm) (Varian) and compounds eluted isocratically at a flow rate of 0.8 ml/min with

5% aqueous methanol. Compounds were detected using dual UV (254nm)

(Knauer) and fluorescence (excitation 350/emission 450) (Shimadzu) detectors

with a Rainin/Varian Liquid Chromatograph. In order to quantify the amount of

pterin in crude extracts, samples were oxidized as described by Milstein and

Kaufman (1989). In short, to 100 µl of extracts 20 µl of 2M H3PO4/ 3M HClO4

mixture was added. Precipitated protein was discarded and the supernatant

incubated for ten minutes at room temperature with ~5 mg of MnO2. The MnO2

was then pelleted out, and the supernatant filtered through a 0.45 µm filter before

analysis. Since oxidized pterins have similar conjugated ring structures, which

impart similar fluorescence intensity, the standard curve obtained with biopterin

was used for quantification of monapterin and neopterin as well (Fig. 4).

18

Figure 4. Standard curve (y = 55391.561x + 163.655) for determination of

oxidized pterins by fluorescence high pressure liquid chromatography.

19

0

25000

50000

75000

100000

125000

Rel

ativ

e Pe

ak A

rea

0 0.5 1 1.5 2 2.5Pterin (µΜµΜµΜµΜ)

20

Spectrophotometric analysis

Pterin spectra were recorded on a Perkin-Elmer Lambda-2

spectrophotometer. The spectra of biological pterins were compared to standards

prepared and stored under argon to prevent autooxidation.

Liquid chromatography//Mass spectroscopy analysis

LC/MS characterization of pterins was conducted on Agilent Technologies

Model G2445 instrument with assistance from Dr. B. Venables. Biological

compounds were separated by reverse phase chromatography and spectra

compared with those of authentic standards

Materials and Supplies

Partially purified Pf11764 CNO enzyme (71-fold activity enrichment) used

for some of the assays was obtained from R. Fernandez. L-Biopterin, 7,8-

dihydro-L-biopterin (H2biopterin), (6R,S)-5,6,7,8-tetrahydro-L-biopterin

(H4biopterin), L-monapterin, 7,8-dihydro-L-monapterin (H2monapterin), (6R,S)-

5,6,7,8-tetrahydro-L-monapterin (H4monapterin), D-neopterin, and 7,8-dihydro-D-

neopterin (H2neopterin) were all purchased from B. Shirck’s Laboratory (Jona,

Switzerland). For some experiments, H4biopterin and biopterin were obtained

from Sigma/Aldrich. 6-Methyl-5,6,7,8-tetrahydropterin (H46-methylpterin) was

obtained from Fluka. The pterin stock solutions were prepared under argon and

concentrations determined spectrophotometrically using published molar

21

extinction values (Pfleriderer, 1985). KCN (97%) was obtained form Aldrich.

K14CN (1.9 GBq/mmol) and sodium [14C]formate (2.1 GBq/mmol) were

purchased from Sigma. All cyanide solutions were prepared immediately prior to

use and were handled according to Environmental Protection Agency and Texas

Commission on Environmental Quality standards.

22

CHAPTER III

RESULTS

Part I. CNO from P. fluorescens NCIMB 11764 Shows a Relaxed Specificity for

Pterin Cofactors

Pf11764 produce pterins of the biopterin, monapterin and neopterin series

Studies by R. Fernandez (Kunz et al., 2001) showed that

tetrahydrobiopterin could substitute for a low mol wt (<10 kDa) cell fraction (Frac

L) required for CNO activity. The analysis of L by HPLC revealed the presence

of a compound identified as H4biopterin, which was thus assumed to be the

natural cofactor for the enzyme. In addition to H4biopterin several other putative

pterin species were also present as shown in Figure 5. Because information on

the occurrence of pterins in bacteria has received limited attention, it became a

point of interest to identify these additional species and determine also, whether

they could satisfy the Pf11764 cofactor requirement. A review of the literature

on the fluorescence character of pterins indicated that the oxidation state has a

marked influence on fluorescence intensity; that is, the more reduced forms

showing decreasingly less fluorescence (Lunte and Kissinger, 1982; Werner et

23

Figure 5. HPLC analysis of the filtrate (10 µl) obtained after passage of Pf11764

HSS through a 10 kDa cutoff ultraconcentrator. Peak identities by comparison

with authentic standards are as follows: a, H2neopterin; b, H2monapterin; c,

H2biopterin; d, threo-H2biopterin.

*This figure was published (Fernandez et al., 2004) and is used in compliance

with the rights provided to the authors by American Society for Microbiology.

24

25

al., 1996). This raised some doubt whether the compound initially identified in

L was, in fact, H4biopterin given that this species shows little fluorescence. When

the peak assumed to be H4biopterin (peak c, Fig. 5) was analyzed by UV

spectroscopy, it gave the spectrum not of H4biopterin but rather that published for

the dihydro derivative (H2biopterin) (λmax 279 and 330 nm) (Fig. 6) (Pfleriderer,

1985). When an authentic standard supplied by Sigma was analyzed

spectrophotometrically it too was found to give the spectrum of H2biopterin, not

H4biopterin. Because the fully reduced pterins are known to be susceptible to

autooxidation (Pfleriderer, 1985), it was concluded that Pf11764 probably

synthesizes the H4biopterin species, but after storage the H2biopterin

predominates. The occurrence of H2biopterin in an authentic standard provided

as H4biopterin, from Sigma, presumably can be explained similarly. In part

because of the doubtful authenticity of the Sigma-supplied sample, standards

hereafter were obtained from the Schirck’s Laboratory in Jona, Switzerland.

H2biopterin from this laboratory gave the same UV spectrum as the biological

compound.

The occurrence of H2biopterin in L raised the question can it also serve as

a cofactor for CNO? Given that L samples stored for some time showed no

appreciable loss in their ability to complement proteins in H and contained,

presumably, H2biopterin, it was hypothesized that H2biopterin might support

activity. To test this, both authentic H4biopterin and H2biopterin obtained from the

26

Figure 6. UV spectra of biologically produced H2biopterin and commercial

standards of H2biopterin and H4biopterin. The samples (25 µM) were analyzed in

mobile phase (5% methanol) at pH 7.

27

28

Schirck’s laboratory were supplied to high mol wt protein fractions (H) of Pf11764

and CNO activity determined by monitoring cyanide conversion to CO2. As

shown in Figure 7, both cofactors were equally capable of supporting CNO

activity. The above findings raised interesting questions regarding CNO and the

oxidation state of the needed cofactor since the classical pterin-dependent

hydroxylases, AAH and NOS, display a rather strict requirement for H4biopterin

(Kappock and Caradonna, 1996; Alderton et al., 2001; Marletta et al., 1998).

Emphasis on identification of the other putative pterins present in fraction L (Fig.

5) took on increasingly more importance since it was hypothesized that perhaps

these compounds could also support enzymatic activity. A comparison of the

elution times with those of standards from Shirck’s laboratory showed that the

peak eluting at 5.5. min was the same as H2neopterin and the peak at 6.5 min

coeluted with H2monapterin. Another peak, at 11.0 minutes, was identified as the

threo isomer of H2biopterin (vs the more abundant erythro isomer) by comparing

its elution time with that published for a standard chromatographed under the

same conditions (Fukushima and Nixon, 1980). The identities of H2neopterin

and H2monapterin were further confirmed by LC/MS in collaboration with Dr. B.

Venables each giving expected H + 1 molecular ions of 256. Collectively, these

data indicated that Pf11764 synthesizes at least four separate pterin species

including, H2biopterin, threo-H2biopterin, H2monapterin, and H2neopterin all of

which probably arise from autooxidation of their fully reduced counterparts.

29

Figure 7. Effect of biopterins on cyanide oxidation activity by cell extracts of

Pf11764. Assays were carried out for 15 min at 30°C in 0.25 ml of Na-K buffer

containing high mol wt protein fraction (Fraction H) (1 mg), 4 mM NADH, 0.1 mM

K14CN and 0.5 µM reduced pterin where necessary.

30

H4B H2B None 0

15

30

45

60

75

90%

Con

vers

ion

of K

14C

N to

14C

O2

� � �

% C

onve

rsio

n of

K14

CN

to 14

CO

2

31

Cofactor activity of pterin compounds in Pf11764

Having shown that Pf11764 produced several different pterins,

experiments aimed at determining whether these compounds might also serve as

cofactors were performed. For these purposes, cofactors were supplied to

partially purified CNO from Pf11764 which converts cyanide stoichiometrically to

formate and ammonia. The effect of added pterin on both the rate of substrate

disappearance and ammonia production was determined. Table 1 shows that

both the fully (tetrahydro) and partially reduced (dihydro) forms of monapterin,

neopterin, and biopterin were all capable of enhancing CNO activity at low

cofactor concentrations (0.5 µM). In contrast, the fully oxidized species, showed

no such activity. Also, no activity in the absence of added reduced pterins or in

controls supplied pterin but not protein was observed.

32

Table 1. CNO-enchancing activity of pterin compounds in Pf 11764

mU/mg proteinb

Pterin provideda Cyanide degraded Ammonia formed

H4biopterin 494 + 92 421 + 67

H2biopterin 501 + 38 508 + 88

Biopterin <10 <10

H4monapterin 407 + 38 411 + 11

H2monapterin 444 + 32 379 + 85

Monapterin <10 <10

H2neopterin 412 + 36 410 + 61

Neopterin <10 <10

H46-methylpterin 425 + 21 385 + 17

None <10 <10

aAbbreviations are as described in the Materials and Methods.

bReaction mixtures contained 50 µM KCN, 10 µg of CNO-enriched protein

per ml, and 0.5 µM pterin. After 10 minutes the amount of remaining

cyanide and ammonia accumulated were determined. The means and

average deviation from the means for three separate determinations are

shown.

* This data was published (Fernandez et al., 2004) and is used in

compliance with the rights provided to the authors by American Society for

Microbiology.

33

Part II. Cyanide Oxidation in P. putida BCN3 Involves a CNO-mediated

Enzymatic Mechanism

Recovery of cyanide oxidation activity in PpBCN3 cell-free extracts

Previous studies established that cells of Pf11764 induced for cyanide

oxidation activity could be obtained by exposing late stationary-phase cells grown

in glucose-ammonia minimal medium to low cyanide concentrations (0.1 mM)

(Kunz et al., 1994). This greatly simplified acquiring cells induced for enzyme

activity which was recovered in the cytosolic fraction of cells referred to as the

high-speed (150,000 x g) supernatant (HSS). When PpBCN3 was grown

similarly and the cytosolic cell fraction (HSS) assayed for CNO activity by

measuring the rate of cyanide disappearance, activity was also detected. Figure

8 shows that KCN (150 �M) supplied to HSS was completely consumed in 6-8

minutes with the initial rate of cyanide disappearance being linear for about the

first 2.5 minutes. In contrast, when reactions were made anaerobic (argon flush),

or NADH was omitted, no cyanide was consumed. These results suggested that

cyanide degradation in PpBCN3 also occurs by a CNO –mediated mechanism.

Figure 8 further shows that cyanide was not consumed by uninduced cell

extracts, which is consistent also with results showing that CNO in Pf11764 is an

inducible enzyme activity. To further determine whether cyanide was converted

to CO2, HSS were incubated with radiolabelled K14CN and its conversion to

14CO2 determined; the production of CO2 serving further to indicate a similar

34

Figure 8. Time course of cell - free cyanide consumption by P. putida BCN3.

Reaction mixtures (125 or 250 µl) contained NADH (1 mM), HSS (4 mg/ml

protein), and 150 µM KCN. Reaction conditions: �, oxygen and NADH both

present; �, oxygen absent, NADH present; �, oxygen present, NADH absent;

�, uninduced HSS of BCN3.

35

36

pathway of oxidation as determined for Pf11764. As shown in Figure 9,

approximately 80% of the radiolabelled K14CN supplied HSS was recovered as

14CO2 after incubation with induced but not with uninduced cells. The presence

of a CNO-related enzymatic system for cyanide oxidation in PpBCN3 was thus,

strongly inferred. Because previous studies with Pf11764 showed that cells

exposed to cyanide were not only induced for CNO but also NAD+-linked FDH

activity (Kunz et al., 1994; Fernandez, 2004), PpBCN3 cells were tested also for

the induction of FDH activity by determining the extent of 14C-formate conversion

to 14CO2. The presence of FDH activity in PpBCN3 was confirmed by an

approximate 80% recovery of the substrate as CO2. However, formate was

oxidized both by induced and uninduced cells indicating that in PpBCN3 (in

contrast to Pf11764) FDH is constitutively expressed.

Cyanide oxidation by fractionated P. putida BCN3 cell extracts

Studies with PpBCN3 cell extracts were made difficult by two complicating

factors one being the high content of lipid and the second the high level of

endogenous NADH oxidase activity. The former complicated enzyme

fractionation of extracts and the latter the assay of CNO activity by conventional

cyanide-dependent consumption of oxygen and NADH. To overcome this

problem, HSS (or fraction H) proteins were clarified of lipid and unrelated

proteins by binding proteins required for cyanide oxidation to an anion-exchange

support as described in material and methods. The recovered proteins (CNO

37

Figure 9. Cyanide and formate conversion to CO2 by cell extracts of PpBCN3.

Reaction mixtures were incubated for 15 minutes and contained in 0.25 ml of Na-

K buffer the following components: 1 mg of protein, 1 mM NADH or NAD+, and

0.1 mM K14CN or Na[14C]formate. (I, induced; U, uninduced)

38

0

25

50

75

100%

Con

vers

ion

of K

14C

N/[

14C

]For

mat

e to

14C

O2

A B C D

I U I U

K14CN [14C]formate

% C

onve

rsio

n of

K14

CN

or

[14C

]For

mat

e to

14C

O2

39

and FDH) with activity were referred to as the Sepharose (S) fraction. Both H

and S retained the ability to oxidize cyanide as evidenced by the conversion of

cyanide to 14CO2 in 80% yield (Table 2). Analysis of reaction mixtures supplied

Fraction S further revealed the presence of ammonia, which accumulated in

amounts approaching that of CO2. It was therefore concluded that like Pf11764,

the major reaction products of cyanide oxidation by PpBCN3 were CO2 and NH3.

Whereas it was not possible to assay PpBCN3 HSS or fraction H directly for

CNO because of high endogenous levels of NADH oxidase, Figure 10 shows that

when cyanide was added to S, an immediate stimulation in both O2 and/or NADH

uptake occurred. The stoichiometry of uptake was further shown to be

consistent with that expected for CNO; that is 1 mole equivalent of O2 and NADH

each being consumed per mole cyanide provided. Taken together these

results provided strong evidence that substrate oxidation in PpBCN3 also

involves a related CNO-mediated mechanism. However, both H and S fractions

conferred CNO activity without having to be supplemented with pterin cofactor.

These observations were interpreted as indicating either that (i) the PpBCN3

enzyme functions independent of pterin, or (ii) that sufficient pterins remain

present in H or S (despite attempts to remove soluble cofactors) to support

enzyme activity. To investigate the latter possibility, extracts of PpBCN3 were

analyzed for pterin compounds in the manner described for Pf11764.

40

Table 2. Products of cyanide metabolism by cell-free extracts of P. putida BCN3.

PpBCN3 KCN Products formed (µM)

fractiona consumed (µM) CO2b Ammonia

H 100 72 + 7 ND

S 100 80 +3 66 + 6

aReaction mixtures (0.125 ml) contained 0.5 mg protein (H or S), 1 mM NADH,

and 0.1 mM KCN in Na-K buffer.

bCO2 determinations represent theoretical conversion estimates based on results

obtained from radiolabelling experiments.

41

Figure 10. Cyanide oxidation activity measured as NADH oxidation and O2

consumption. Reaction mixtures (0.5 ml) in Na-K buffer contained 1 mg of

Sepharose Q enriched protein and 0.2 mM NADH.

42

43

P. putida BCN3 synthesizes pterins of the biopterin, monapterin and neopterin

series at levels approximately seven times that of Pf11764

Analysis of PpBCN3 cell extracts by HPLC (Fig. 11) revealed, the

presence of the same four pterin species (H2monapterin, H2neopterin,

H2biopterin and threo-H2biopterin) identified also in Pf11764. However,

preliminary analysis suggested that the amounts of these compounds were

significantly higher than that present in Pf11764. This was further

substantiated when the total content of pterins (sum of all four of the above) in

each organism was determined following oxidation to their most stable (and most

fluorescent) oxidized forms. When calculated based on internal cell

concentration, the total amount of pterins in PpBCN3 HSS (as shown in Table 3)

was about seven times that for Pf11764 HSS. This translates to an estimated

internal concentration of 0.26 mM for PpBCN3 versus 0.038 mM for Pf11764. It

can be seen that fraction H also retained a significantly higher amount of pterin

(265 vs. 37 pmol/mg for Pf11764 and BCN3, respectively), thus

helping to explain why H may have retained activity in the absence of any

exogenously added pterin. However, this may not represent the total

explanation since fraction S which contained only 35 pmol/mg pterin (which could

not be removed by extensive dialysis) still displayed activity. It may be noted that

no significant differences in the amount of pterins in extracts of cyanide induced

and uninduced cells for both Pf11764 and PpBCN3 were observed.

44

Figure 11. HPLC analysis of oxidized HSS of PpBCN3. Peak identities by

comparison to authentic standards are as follows: a, H2neopterin; b,

H2monapterin; c, H2biopterin; d, threo-H2biopterin.

45

46

Table 3. Pterin content of cyanide-degrading bacteria

Total pterins in fraction Internal

(pmol/mg)a cell concentrationb

Strain HSS H L S (mM)

Pf11764 113 + 28 37 + 14 1280+112 ND 0.038

PpBCN3 778 + 50 265 + 20 3930 + 220 35+9 0.26

aThe values are means + average deviations of the means for at least two

separate determinations

bCalculated according to Blaunt and Gottschalk (1984) in which one mg of

protein is estimated to be equivalent to an intracellular volume of 3 µl as

determined for most prokaryotes.

47

CHAPTER IV

DISCUSSION

Growth on cyanide as the sole nitrogen source by Pf11764 was previously

shown to require the induction of an enzyme described as CNO responsible for

initiating substrate conversion to ammonia (Kunz et al., 1994; Wang et al., 1996;

Kunz et al., 2001). Subsequent studies demonstrated that CNO displayed the

properties of a pterin-dependent hydroxylase given that H4biopterin could replace

a required cofactor component (Kunz et al., 2001; Fernandez, 2004). This study

demonstrates that not only H4biopterin, but also a number of reduced pterins can

satisfy the CNO cofactor requirement. These were shown to include, for

example, the tetrahydro and diydro forms of monapterin, neopterin, and biopterin

all of which were also identified in cell extracts of both Pf11764 and PpBCN3.

The detection of H2monapterin in both Pf11764 and PpBCN3 finds analogy with

early reports describing this compound as the major pterin in two different

Pseudomonas species (Viscontini and Buhler-Moor, 1968; Guroff and Rhoads,

1969). In one of these, the fully reduced species along with reduced

H4biopterin were believed to serve as natural cofactors for phenylalanine

hydroxylase (Guroff and Rhoads, 1969).

The broad specificity of the Pf11764 CNO enzyme towards different

structural pterins (e.g., bio-, neo-, monapterin) parallels properties of the AAH

and NOS enzymes (Kappock and Caradonna, 1996; Marletta et al., 1998).

48

However, there are distinct differences between CNO, AAH, and NOS with

regard to the oxidation state of pterin compounds capable of satisfying the

cofactor requirement. For example, AAH and NOS each display a rather strict

requirement for the fully reduced tetrahydropterins whereas CNO appears to

function equally as well with either the tetrahydro or dihydro derivatives.

Interestingly, all three enzymes share an inability to utilize the fully oxidized

pterins. In addition, as demonstrated also for NOS, the amount of pterin

needed by CNO was very small (saturating at 0.5 �M), and therefore, it is

assumed to function catalytically.

The investigation of cyanide oxidation by P. putida BCN3 revealed that the

main products of cyanide breakdown were NH3 and CO2. Further studies showed

that substrate conversion required O2 and NADH each consumed in a 1:1:1 ratio

to cyanide provided. These three characteristics closely resemble those of

Pf11764 CNO system thus leading to the conclusion that an enzyme with

oxygenase character is at work in this organism as well. Given that cyanide is

metabolized by a similar pathway as that described for Pf11764, we

hypothesized that a similar requirement for reduced pterins by the PpBCN3 CNO

enzyme might also exist. However, because of the high pterin content of

PpBCN3 this requirement was difficult to demonstrate. Nonetheless, a

dependency on pterins for activity cannot totally be excluded for the following

reasons. First, based on the estimated amount of protein-bound pterin in

49

PpBCN3, the pterin concentration in reaction mixtures supplied fraction S can be

calculated to be approximately 140 nM (in reactions supplied 4 mg/ml protein).

Interestingly, this concentration is similar to the estimated Km values for pterin

described for neuronal NOS (30 nM) (Adak et al., 1999) and NOS from Bacillus

subtilis (100 nM) (Adak et al., 2002) giving cause to believe that the amounts

present may in fact be sufficient for activity. Second, some experiments (data

not shown) gave evidence of an influencing effect of pterin on enzymatic activity.

For example, when reaction mixtures were supplemented with H2biopterin, the

amount of CO2 recovered was slightly elevated above that observed in its

absence (e.g., 85% vs. 72% respectively) (data not shown). Also, when the

H2biopterin provided to S-containing reaction mixtures was increased from the

usual 0.5 �M to 50 �M, cyanide oxidation was completely inhibited. Importantly,

this observation finds analogy with the properties of both AAH and NOS in which

case an excess of reduced pterin reportedly also inhibits these enzymes

(Kappock and Caradonna, 1996).

While the role of pterins as cofactors for CNO-catalyzed processes

remains to be determined, recent studies by R. Fernandez (2004) showing that

the Pf11764 enzyme is in fact, an aggregate of proteins that catalyzes substrate

conversion by a potential radical mechanism parallel the properties of NOS

enzymes. For example, in the case of NOS, H4biopterin appears to play a role

both in the association of large subunits (130 kDa) that make up the structure of

50

NOS and as a radical transfer agent (Wei et al., 2003). Thus, the overlap in

properties of the two enzymatic systems gives reason to believe that reduced

pterins may function similarly in each.

In summary, strong evidence that cyanide oxidation occurs by related

oxygenolytic enzymatic mechanisms in two separate cyanide-degrading bacteria

has been provided. These findings serve to indicate that the mechanisms

evolved by microorganisms for cyanide detoxification and assimilation in the

environment are probably more related than previously recognized.

51

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