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Journal of Experimental Botany, Vol. 59, No. 7, pp. 15431554, 2008

doi:10.1093/jxb/ern104 Advance Access publication 9 April, 2008

SPECIAL ISSUE REVIEW PAPER

RuBisCO-like proteins as the enolase enzyme in the

methionine salvage pathway: functional and evolutionary

relationships between RuBisCO-like proteins andphotosynthetic RuBisCO

Hiroki Ashida1,*, Yohtaro Saito1, Toshihiro Nakano1, Nicole Tandeau de Marsac2, Agnieszka Sekowska3,

Antoine Danchin4 and Akiho Yokota1

1 Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences, 8916-5 Takayama,

Ikoma, Nara, 630-0101 Japan2 Unitedes Cyanobacteries, CNRS URA 2172, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15,France3 Unitede Genetique in silico, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France4 Genetics of Bacterial Genomics, CNRS URA 2171, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex15, France

Received 30 January 2008; Revised 26 February 2008; Accepted 28 February 2008

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase

(RuBisCO) is the key enzyme in the fixation of CO2 in

the Calvin cycle of plants. Many genome projects have

revealed that bacteria, including Bacillus subtilis,

possess genes for proteins that are similar to the largesubunit of RuBisCO. These RuBisCO homologues are

called RuBisCO-like proteins (RLPs) because they are

not able to catalyse the carboxylase or the oxygenase

reactions that are catalysed by photosynthetic

RuBisCO. It has been demonstrated that B. subtilis

RLP catalyses the 2,3-diketo-5-methylthiopentyl-

1-phosphate (DK-MTP-1-P) enolase reaction in the

methionine salvage pathway. The structure of

DK-MTP-1-P is very similar to that of ribulose-

1,5-bisphosphate (RuBP) and the enolase reaction is

a part of the reaction catalysed by photosynthetic

RuBisCO. In this review, functional and evolutionary

relationships between B. subtilis RLP of the methio-

nine salvage pathway, other RLPs, and photosynthetic

RuBisCO are discussed. In addition, the fundamental

question, How has RuBisCO evolved? is also consid-

ered, and evidence is presented that RuBisCOs

evolved from RLPs.

Key words: Bacillus subtilis, CO2 fixation, 2,3-diketo-5-

methylthiopentyl-1-phosphate enolase, methionine salvage

pathway, molecular evolution, photosynthesis, RuBisCO,

RuBisCO-like protein.

Introduction

Ribulose-1,5-bisphosphate carboxylase/oxygenase

(RuBisCO) is the key enzyme in the Calvin cycle.

RuBisCO catalyses the carboxylase reaction that fixes

CO2 on RuBP and produces two molecules of

3-phosphoglycerate (Andrews and Lorimer, 1987; Hartman

and Harpel, 1994; Roy and Andrews, 2000). The

carboxylase reaction is the initial reaction in the Calvin

cycle, and fixed CO2 is utilized as the carbon source to

synthesize sugars and/or starch for the growth of photo-

synthetic organisms. Nevertheless, RuBisCO has ineffi-

cient enzymatic properties (Andrews and Whitney, 2003).

In particular, RuBisCO catalyses an oxygenase reaction

that fixes O2 into RuBP, and this reaction antagonizes thecarboxylase reaction in atmospheres in which O2 is much

more abundant than CO2 (Andrews and Lorimer, 1987;

Hartman and Harpel, 1994; Roy and Andrews, 2000).

The oxygenase reaction is the starting reaction of

* To whom correspondence should be addressed. E-mail: [email protected]

The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]


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photorespiration, which releases CO2 and NH3 and wastes

energy. In addition, the turnover of the carboxylasereaction by RuBisCO is a few to several times per second,

even when substrates are saturating (Andrews and

Whitney, 2003). Therefore, the photosynthetic CO2assimilation rate in plants can be limited by RuBisCO

(Hudson et al., 1992; Von Caemmerer et al., 1997). Plants

manage to perform photosynthetic CO2 fixation byaccumulating large amounts of RuBisCO protein to

compensate for the inefficient properties of RuBisCO.

Indeed, the RuBisCO protein constitutes ;50% of the

soluble proteins in plant leaves and is the most abundant

protein on earth (Ellis, 1979). Given these observations,

RuBisCO is the critical target for the improvement of

photosynthetic efficiency and productivity of plants.

Manipulation of RuBisCO to increase the carboxylation

rate and/or achieve high CO2 and low O2 affinity would

promote the efficiency of photosynthesis (Andrews and

Whitney, 2003).

Why has RuBisCO failed to evolve into an efficient

enzyme that does not catalyse the oxygenase reaction?Has there been an opportunity to evolve a more efficient

enzyme? The answers to these questions should be

resolved by a study of the molecular evolution of

RuBisCO. RuBisCO is classified into three forms, forms

I, II, and III, based on amino acid sequences and protein

structures, and all RuBisCOs in these groups catalyse both

carboxylase and oxygenase reactions. CO2 and O2 are

freely diffusible gasses, and O2 is smaller than CO2,

making it difficult to create a physical barrier to dioxygen

where CO2 has to be present; but why did RuBisCO not

acquire some sort of chemical barrier to prevent the action

of O2? This question cannot be answered by only

considering the evolutionary relationships among the threeforms of RuBisCO. Instead there is a need to understand

the emergence of RuBisCO in terms of its molecular

evolution.

Interestingly, there are proteins that are suitable for the

analysis of the molecular evolution of RuBisCO. Bacteria

and Archaea possess genes encoding proteins that have

considerable sequence identity to the large subunit of

photosynthetic RuBisCO. These proteins cannot catalyseeither the carboxylase or the oxygenase reactions of

RuBisCO, and are named RuBisCO-like proteins (RLPs).

RLPs represent a fourth class of the RuBisCO enzyme

family (Hanson and Tabita, 2001). Among the Bacteria

and Archaea, the function of RLP has been revealed onlyin Bacillus subtilis. Bacillus subtilis RLP catalyses the2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P)

enolase reaction, which is the fourth step in the methionine

salvage pathway (Ashida et al., 2003). Bacillus subtilisRLP catalyses an analogous reaction to photosynthetic

RuBisCO since its substrate resembles the structure of

RuBP, rationalizing the tight linkage between RLPs and

RuBisCO.

Until recently, the molecular evolution of RuBisCO had

been discussed using an evolutionary clock involving the

emergence of form I and form II RuBisCOs. It was not

possible to discuss the molecular evolution of RuBisCO

before the enzyme acquired the ability to fix CO2 because

ancestor proteins of RuBisCO had not yet been found.

The finding of RLPs, as a fourth form of the RuBisCO

family of enzymes, has enabled discussion of themolecular evolutionary relationship between RLPs and

RuBisCO. In Guptas hypothesis for the evolution of

Bacteria, low G+C Gram-positive bacteria, including

B. subtilis, were predicted to be the most ancient bacteria,which emerged near the root of the phylogenetic tree of

life. Considering the molecular evolution of RuBisCO,

based on Guptas hypothesis (Gupta, 1998; Gupta et al.,1999), RLPs may still retain features of the ancestor

protein of RuBisCO, because low G+C Gram-positive

bacteria and Archaea, possessing RLP genes, emerged

before cyanobacteria and photosynthetic bacteria, which

both utilize RuBisCO in the Calvin cycle. Thus, the

discovery of RLPs and determination of RLP functionprovide a new approach to elucidate the evolution of

RuBisCO. However, the methionine salvage pathway, to

recycle reduced sulphur in sulphur-deprived environ-

ments, may not be a more ancient metabolic pathway,

with respect to evolutionary processes. This is because

hydrogen sulphide, a reduced form of sulphur, was

presumably abundant early on Earth when the most

primitive organisms were living with minimal numbers of

genes and metabolic pathways (Delano, 2001). It is

possible, therefore, that an unknown ancestor protein of

RuBisCO already existed before RLPs emerged as the

enolase enzyme in the methionine salvage pathway, and

that RuBisCO has evolved from this ancestor protein,rather than via RLP in the methionine salvage pathway.

Thus, discussion of the evolutionary relationship between

RuBisCO and RLPs in the methionine salvage pathway

still cannot answer such fundamental questions as: Has

RuBisCO evolved via RLPs? and What is the origin of

RuBisCO? In this review, functional and evolutionary

relationships between RLPs and photosynthetic RuBisCO

are discussed, and a hypothesis is also proposed for this

fundamental question of RuBisCO evolution.

RuBisCO-like proteins are widely distributed in

Bacteria and in ArchaeaGenome projects have revealed that many of them possess

genes encoding RLPs that show similarity to the large

subunit of RuBisCO. In a phylogenetic tree, produced

using deduced amino acid sequences from the large

subunits of RuBisCO and RLPs, RuBisCOs are classified

into three forms, forms I, II, and III (Fig. 1) (Hanson and

Tabita, 2001; Ashida et al., 2003, 2005). Form I consistsof eight large and eight small subunits of 5055 kDa and

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1218 kDa, respectively, and is distributed widely among

photosynthetic organisms such as plants, algae, cyanobac-

teria, and photosynthetic and chemoautotrophic proteo-

bacteria (Tabita, 1999). Form II is composed only of large

subunits and is found mainly in some photosynthetic

proteobacteria and chemoautotrophic bacteria (Tabita,

1999). Form III is composed of only large subunits and is

found in Archaea (Tabita, 1999). On the other hand, RLPs

form three clades, groups a, b, and c, that are differentfrom the clades of photosynthetic RuBisCO (Fig. 1).

Group a includes RLPs from Bacillus species, thecyanobacterium Microcystis aeruginosa, the sulphate-reducing Archaeon Archaeoglobus fulgidus, and thephotosynthetic bacteria Rhodopseudomonas palustris and

Rhodospirillum rubrum. Group b contains RLPs fromnon-photosynthetic proteobacteria Palaromonas sp.,

Fig. 1. Phylogenetic tree constructed from the amino acid sequences of the large subunits of RuBisCO and of RLPs. The multiple sequencealignment and the phylogenetic tree were produced with CLUSTALW and TREE VIEW. Full names of species are as follows: B. subtilis, Bacillussubtilis; B. licheniformis, Bacillus licheniformis; Bacillus sp., Bacillus sp. NRRL B-14911; B. thuringiensis, Bacillus thuringiensis; B. anthracis,Bacillus anthracis; B. weihenstephanesis, Bacillus weihenstephanesis; B. cereus, Bacillus cereus; G. kaustrophilus, Geobacillus kaustrophilus;

B. clausii, Bacillus clausii; E. sibiricum, Exiguobacterium sibiricum; Lyngbya sp., Lyngbya sp. PCC8106; M. aeruginosa, Microcystis aeruginosaPCC7806; A. fulgidus, Archaeoglobus fulgidus; H. mobilis, Heliobacillus mobilis; O. tauri, Ostreococcus tauri; O. granulosus, Oceanicolagranulosus; N. mobilis, Nitrococcus mobilis; H. halophila, Halorhodospira halophila; R. palustris, Rhodopseudomonas palustris; R. rubrum,Rhodospirillum rubrum; Aurantimonas, Aurantimonas sp. SI85-9A1; F. pelagi, Fulvimarina pelagi; Polaromonas sp., Polaromonas sp. JS666;C. salexigens, Chromohalobacter salexigens; P. putida, Pseudomonas putida; D. acidovorans, Delftia acidovorans; B. bronchiseptica, Bordetellabronchiseptica; M. loti, Mesorhizobium loti; S. meliloti, Sinorhizobium meliloti; A. aurescens, Arthrobacter aurescens; P. luteolum, Pelodictyonluteolum; C. chlorochromatii, Chlorobium chlorochromatii; C. phaeobacteroides, Chlorobium phaeobacteroides; P. aestuarii, Prosthecochlorisaestuarii; C. tepidum, Chlorobium tepidum; R. sphaeroides, Rhodobacter sphaeroides; T. denitrificans, Thiobacillus denitrificans; D. aromatica,Dechloromonas aromatica; N. hamburgensis, Nitrobacter hamburgensis; S. WH8102, Synechococcus sp. WH8102; P. marinus, Prochlorococcusmarinus; N. punctifome, Nostoc punctiforme PCC73102; T. elongatus, Thermosynechococcus elongatus; S. PCC6803, Synechocystis sp. PCC6803; S.elongatus, Synechococcus elongatus PCC6301; R. sphaeroides, Rhodobacter sphaeroides; R. gelatinosus, Rubrivivax gelatinosus; B. japonicum,Bradyrhizobium japonicum; M. jannaschii, Methanocaldococcus jannaschii; M. barkeri, Methanosarcina barkeri; M. mazei, Methanosarcina mazei;M. acetivorans, Methanosarcina acetivorans; T. kodakarensis, Thermococcus kodakarensis; H. butylicus, Hyperthermus butylicus; N. pharaonis,Natronomonas pharaonis; P abyssi, Pyrococcus abyssi; P. horikoshii, Pyrococcus horikoshii; P. furiosus, Pyrococcus furiosus; M. marisnigri,Methanoculleus marisnigri.

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Bordetella bronchiseptica, and Mesorhizobium loti. RLPs

from green non-sulphur bacteria of the genus Chlorobiumand the proteobacteria Rhodopseudomonas sp. are classi-fied in group c.

Comparing amino acid sequences between B. subtilisRLP and photosynthetic RuBisCO, B. subtilis RLP shows

;23% identity to form I and II RuBisCOs and ;30%

identity to form III RuBisCOs. In the same group a, B.subtilis RLP shows 78.5, 62.0, 59.4, 22.7, 35.6, 34.8,23.2, and 22.7% identity to RLPs of Bacillus lichen-iformis, Geobacillus kaustophilis, Bacillus anthracis,

Bacillus clausii, M. aeruginosa, A. fulgidus, R. rubrum,and R. palustris, respectively. On the other hand, B.subtilis RLP shows 27.5, 26.6, and 23.5% identity to

RLPs in group b, from Palaromonas sp., B. bronchisep-tica, and M. loti, respectively, and 25.1% and 26.8%identity to RLPs in group c, from R. palustris and

Chlorobium tepidum, respectively. Considering this distri-

bution and in the absence of horizontal gene transfer,

RLPs should have evolved widely, because the three

clades of RLPs show a greater distance from each other

compared with that with each form of RuBisCO (Fig. 1).

Using X-ray crystallography and point mutational

analysis, Hanson and Tabita (2001) proposed 19 amino

acid residues of photosynthetic RuBisCO as essential for

catalysis (Fig. 2). Alignment of RLPs sequences fromRLPs shows conservation of 918 of the 19 residues.

Bacillus subtilis RLP has 11 conserved residues (Fig. 2).These observations indicate that RLPs are very unlikely to

catalyse the carboxylase and oxygenase reactions that are

catalysed by photosynthetic RuBisCO.

What is the function of RLPs? Is there a functional and

evolutionary relationship between RLPs and photosyn-

thetic RuBisCO? The answers to these questions have

interested us with respect to the molecular evolution of

RuBisCO.

Fig. 2. Partial multiple sequence alignment of the large subunits of RuBisCO and RLPs. Sequences flanking the residues involved in the carboxylasereaction catalysed by RuBisCO are aligned. Active site residues conserved in RuBisCO are shown in grey letters on a black background. Residuesinvolved in catalysis and enolization of RuBP are indicated above the alignment by C and E, respectively. Lys201 for carbamylation is indicated by*. 1 and 5 above the alignment indicate residues involved in binding of the phosphate groups on C1 and C5 of RuBP, respectively. White colouredletters indicate residues that are not conserved in RLPs. The alignment is numbered according to the sequence of the large subunit of spinachRuBisCO.

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The road to identification of the function ofB. subtilis RLP

In order to identify the function of RLP, the B. subtilisgenome was explored when it was completed. When the

analysis began, the gene encoding RLP in B. subtilis wasannotated as a gene of unknown function, now named

mtnW, coding for a protein similar to the large subunit of

RuBisCO. It was initially predicted that B. subtilis RLPcould not catalyse the carboxylase and oxygenase reactions

of photosynthetic RuBisCOs, because this RLP has only

11 of 19 amino acid residues that are essential for this

catalysis (Fig. 2). In order to confirm this prediction, an

analysis was carried out to determine whether B. subtilisRLP can catalyse the carboxylase reaction using recombi-

nant protein expressed in Escherichia coli. Indeed,

B. subtilis RLP showed no carboxylase activity. This resultclearly showed that B. subtilis RLP functions as an enzymein a different metabolic pathway from that of RuBisCO.

The mtnW gene for the RLP of B. subtilis is the firstgene of the mtnWXBD operon (Fig. 3a). The mtnKAoperon lies in the vicinity of the mtnWXBD operon

(Fig. 3a). It has been predicted that both operons have

a leader mRNA, the S box, known to regulate the

expression of the genes involved in methionine metabo-

lism (Murphy et al., 2002; Sekowska and Danchin,

2002) (Fig. 3a). The S box is a riboswitch that regulates

gene expression via transcription termination (Winkler

et al., 2003; Montange and Batey, 2006), and enhances

the expression of target genes in response to methioninestarvation by detection of the concentration of S-

adenosylmethionine, a metabolite indicator for methionine

levels in vivo. In order to analyse the response of the

mtnWXBD operon to methionine starvation, lacZ was

inserted into the internal region of mtnW. Analysis of the

expression of the mtnWXBDlacZ operon showed that

expression of lacZ was not induced on growth medium

containing methionine but was induced on medium

containing NH4Cl, glutamine, and serine (Fig. 4a). In

addition, lacZ expression was enhanced by removing

methionine from the culture medium after pre-culture on

medium including methionine (Fig. 4b). These results

clearly suggested that this operon was involved in

Fig. 3. The methionine salvage pathway and related operons in B. subtilis. The mtnWXBD and mtnKA operons consist of genes involved in themethionine salvage pathway. The expression of genes in both operons is regulated by S boxes. (b) The methionine salvage pathway in B. subtilis.



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methionine metabolism. Furthermore, in mtnWXBD and

mtnKA operons, MtnD showed high similarity to1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase,

and MtnK has been identified as the methylthioribose

(MTR) kinase (Sekowska et al., 2001), suggesting thatboth operons function in the methionine salvage pathway

(Fig. 3b) (Grundy and Henkin, 2002; Murphy et al., 2002;Sekowska and Danchin, 2002). These data indicate that

B. subtilis RLP would catalyse a reaction step somewhere

in this pathway. Many organisms, including bacteria

(Sekowska et al., 2000; Grundy and Henkin, 2002), yeast(Marchitto and Ferro, 1985), plants (Burstenbinder et al.,

2007), and mammals (Wray and Abeles, 1995; Garcia-

Castellano et al., 2002), utilize the methionine salvagepathway to recycle organic sulphur from MTR. In this

pathway, organic sulphur is salvaged from MTR, which is

produced from methylthioadenosine (MTA), a waste prod-

uct of polyamine (Sekowska et al., 2000; Grundy andHenkin, 2002), mugineic acid (Ma et al., 1995), and

ethylene synthesis (Burstenbinder et al., 2007). In themethionine salvage pathway that has been proposed in

Klebsiella, MTR is phosphorylated to MTR-1-phosphate(MTR-1-P) by the MTR kinase, and then MTR-1-P

is isomerized by an isomerase to methylthioribulose-1-

phosphate (MTRu-1-P) (Saito et al., 2007). MTRu-1-Pundergoes a dehydration that is catalysed by a dehydratase

and produces DK-MTP-1-P (Furfine and Abeles, 1988;Ashida et al., 2003, 2005, 2007; Sekowska et al., 2004).DK-MTP-1-P is converted to 1,2-dihydroxy-3-keto-5-

methylthiopentene (DHK-MTPene), via the intermediate,

2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate (HK-

MTPenyl-1-P), by a bi-functional enzyme, enolase/phos-

phatase (Balakrishnan et al., 1993; Myers et al., 1993). In

the final step, DHK-MTPene is converted to formate and

2-keto-4-methylthiobutyrate (KMTB) by a dioxygenase

(Wray and Abeles, 1993), and KMTB is transaminated to

methionine (Berger et al., 2003). In Klebsiella, each stephas been predicted based on the analysis of metabolites,

but this pathway remains to be fully understood. The

structures of MTRu-1-P and of DK-MTP-1-P are similar tothe structure of RuBP, and D-glycero-2,3-pentodiulose-

1,5-bisphosphate, is a by-product of the oxygenase re-

action catalysed by RuBisCO (Pearce and Andrews, 2003).

It is therefore predicted that B. subtilis RLP is MTRu-1-Pdehydratase or DK-MTP-1-P enolase/phosphatase.

Growth assays were used to examine whether RLP was

involved in the methionine salvage pathway, using an

RLP-deficient mutant of B. subtilis and pathway metabo-lites. The wild type grew in the culture medium contain-

ing methionine, MTA, or KMTB as a sole source of

sulphur (Fig. 5a, b). The RLP-deficient mutant grew in

culture medium containing methionine or KMTB, but not

in medium containing MTA as a sole source of sulphur(Fig. 5a, b) (Ashida et al., 2003). This result clearly

showed that B. subtilis RLP catalyses a reaction stepbetween MTA and KMTB in the methionine salvage

pathway. To determine this step, enzymes, their sub-

strates, and products in the methionine salvage pathway of

B. subtilis were identified, step-by-step, using recombinantproteins encoded in the mtnWXBD and mtnKS operonsand employing 1H-NMR and UV-VIS spectroscopy(Ashida et al., 2003). All reaction steps and enzymeswere determined in the methionine salvage pathway,

consisting of mtnA, mtnW, mtnX, mtnB, and mtnD genes,which encode MTR-1-P isomerase, DK-MTP-1-P enolase,

HK-MTPenyl-1-P phosphatase, MTRu-1-P dehydratase,and DHK-MTPene dioxygenase, respectively (Fig. 3b)

(Ashida et al., 2003). In this way, B. subtilis RLP wasidentified as the DK-MTP-1-P enolase. The enolase/

phosphatase activities in other organisms were different

from that in the methionine salvage pathway of B. subtilis,in that these steps were catalysed by two enzymes in

B. subtilis, RLP for enolization and HK-MTPenyl-1-Pphosphatase for deposphorylation (Fig. 3b).

Fig. 4. Induction of the mtnWXBD operon. (a) A Bacillus subtilismutant with lacZ inserted into the mtnW gene was grown on culturemedium containing 40 mg ml1 X-gal and 1 mM NH4Cl, glutamine,serine, or methionine. Blue colonies were produced (black colour in thismonochromatic figure) by the expression of a lacZ reporter gene withinthe mtnWXBD operon, when the mtnWXBD operon was induced. (b)The same mutant was grown in Spizizen minimal medium containingmethionine (0.3 mM), split at 0 h, and grown in the presence (opencircles) or absence (filled squares) of methionine (0.3 mM). Cells werecollected every 1 h, and b-galactosidase activities were measured using

the total soluble fraction extracted from cells.

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The function of RLP from the cyanobacterium Micro-cystis aeruginosa PCC7806 was also identified, based onanalysis of B. subtilis RLP. Interestingly, this cyanobacte-

rium possesses genes for both RLP and photosyntheticRuBisCO. The gene for RLP of M. aeruginosa couldrescue an RLP-deficient mutant of B. subtilis when they

were grown in culture medium including MTA as the sole

source of sulphur (Fig. 5c) (Carre-Mlouka et al., 2006).The recombinant M. aeruginosa RLP showed DK-MTP-1-P enolase activity (Carre-Mlouka et al., 2006), suggest-ing that M. aeruginosa RLP is the DK-MTP-1-P enolasein this cyanobacterium. This was the first report that RLP,

functioning in the methionine salvage pathway, co-existed

with a photosynthetic RuBisCO in the same organism. Inaddition, cyanobacteria are considered to be the ancestors

of photosynthetic eukaryotic chloroplasts. These facts are

interesting, with respect to the evolutionary relationship

between RLPs and photosynthetic RuBisCO. Assuming

that RLP, in the methionine salvage pathway, is related to

the ancestor protein of RuBisCO, based on Guptas

bacterial evolution hypothesis, the examination of this

cyanobacterium may provide the missing link between

micro-organisms utilizing RLPs for methionine salvage

and photosynthetic organisms with RuBisCO. However, it

is unclear whether this cyanobacterium has retained the

gene for RLP from an ancestral organism or acquired it

from recent lateral transfer. Further analysis of cyanobac-

teria possessing both genes for RLP and RuBisCO are

needed to settle this question.

What is the comparison of catalysing reactionsbetween B. subtilis RLP and photosyntheticRuBisCO telling us?

It seems that there is no linkage between B. subtilis and

M. aeruginosa RLPs and photosynthetic RuBisCO,because RLPs function in the methionine salvage path-

way, which is far from the Calvin cycle for CO2 fixation.

However, interesting observations can be made about the

catalysing reactions of these RLPs and photosynthetic

RuBisCO. DK-MTP-1-P, a substrate of RLPs, has

structural similarity to RuBP, the substrate for photosyn-thetic RuBisCO, except that in DK-MTP-1-P, the OH

group and proton on C3 of RuBP are replaced by

a carbonyl group (Fig. 6) (Ashida et al., 2005). Addition-ally, in DK-MTP-1-P, the OH group on C4 and the

phosphate group on C5 of RuBP are replaced by a proton

and a methylthio group, respectively (Fig. 6). The

functional similarities between DK-MTP-1-P enolase and

RuBisCO are found not only by comparing substrates, but

also by comparing steps in the catalytic cycle. The

carboxylase reaction, catalysed by RuBisCO, consists of

five sequential reactions: (i) enolization of RuBP by

abstraction of a proton on C3; (ii) carboxylation of C2;

(iii) hydration of C3; (iv) cleavage of the C2C3 bond;and (v) protonation of the C2 of the upper aci-acid formof 3-phosphoglycerate (Mauser et al., 2001). The firstcatalysing reaction, enolization of RuBP, is the same

reaction as enolization of DK-MTP-1-P in RLP, except

that the proton on C1 of DK-MTP-1-P is abstracted in the

RLP-catalysed reaction. Lys175, Lys201, Asp203, and

Glu204 are essential residues for RuBP enolization inRuBisCO (Cleland et al., 1998). In particular, Lys201 is

carbamylated by CO2, and this carbamate is stabilized by

co-ordination of an Mg2+ ion (Andrews and Lorimer,

1987; Hartman and Harpel, 1994; Roy and Andrews,

2000). The carbamylated Lys201, Asp203, and Glu204

are essential residues to bind the Mg2+ ion, whichstabilizes the endiol form of RuBP (Cleland et al., 1998).

In the proposed mechanism for the enolization of RuBP,the carbamylated Lys201 is the base that abstracts the

proton of C3, while the amino group of Lys175 initially

stabilizes the resultant endiol of RuBP and then acts as the

acid that protonates the oxygen anion (Cleland et al.,1998) (Fig. 6). Mutation of these residues leads to a lack

of RuBP enolization activity in RuBisCO (Estelle et al.,

Fig. 5. Rescue of the RLP-deficient mutant of B. subtilis by thegene for photosynthetic RuBisCO or by cyanobacterium RLP. Weused two RLP-deficient mutants of B. subtilis, DRLP1 (mtnW::spcamyE::mtnXBD) (Carre-Mlouka et al., 2006) and DRLP2 (mtnW::pMu-tinT3) (Ashida et al., 2003). Strain DRLP1 was transformed with theexpression vector, pDG148 (Stragier et al., 1998), harbouring the RLPgene from M. aeruginosa PCC7806, yielding DRLP/MaRLP+. The genefor photosynthetic RuBisCO from the photosynthetic bacterium Rhodo-spirillum rubrum was integrated into the amyE gene in DRLP2, givingstrain DRLP/RuBisCO+. Wild type (a), DRLP1 (b), DRLP/MaRLP+ (c),and DRLP/RuBisCO+ (d) were grown for 35 h in Spizizen minimalmedium (Anagnostopoulos and Spizizen, 1961) containing 1 mMisopropyl-b-D-thiogalactopyranoside and 0.5 mM MTA as the solesource of sulphur. When required, spectinomycin, erythromycin,kanamycin, and chloramphenicol were added to a final concentration of100, 0.5, 5, and 5 mg ml1, respectively.



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1985; Hartman et al., 1987; Gutteridge et al., 1988).Interestingly, B. subtilis and M. aeruginosa RLPs con-serve these residues that are predicted to be essential

residues for the DK-MTP-1-P enolase reaction (Fig. 2). It

was easy to predict that Lys201 was also the catalytic

residue for abstraction of a proton in B. subtilis RLP.Recently, Imker et al. (2007) reported the X-ray crystal

structure of Geobacillus kaustophilus RLP, complexedwith an analogue of DK-MTP-1-P, 2,3-diketohexane-1-

phosphate (DK-H-1-P). Geobacillus kaustophilus belongsto the same clade Bacillaceae as B. subtilis. Geobacillus

kaustophilus RLP catalyses the DK-MTP-1-P enolase

reaction and has conserved residues Lys175, Lys201,

Asp203, and Glu204, which are also conserved in

B. subtilis and M. aeruginosa RLPs (Fig. 2). Lys201 wascarbamylated, and carbamylated Lys201, Asp203, and

Glu204 were Mg2+ ligands in the 3D structure of G.kaustophilus RLP (Imker et al., 2007). The arrangementof Lys175, carbamylated Lys201, Asp203, and Glu204 in

the active site was very similar to that in RuBisCO.Therefore, carbamylated Lys201 was located too far from

C1 of DK-H-1-P to abstract a proton, and Lys175 was

also located at a distance from C1. In this RLP, Asn123,

conserved in RuBisCO, was replaced by lysine (Fig. 2),

and Lys123 was appropriately positioned to abstract

a proton from C1 of DK-H-1-P. In fact, the enolase

activity was retained by alanine substitutions for Lys175

and Lys201, but alanine substitution for Lys123 resulted

Fig. 6. Proposed mechanism for the enolization of RuBP by RuBisCO and of DK-MTP-1-P by Bacillus RLP. In enolization of RuBP by RuBisCO,the carbamate group attached to Lys201 is the base that abstracts a proton from C3, while the amino group of Lys175 initially stabilizes the resultantenolate and then acts as the acid that protonates the oxygen anion. In the reaction catalysed by RLP, if RLPs utilize the same residues as RuBisCO,de-protonation at C1 and re-protonation at the oxygen anion are carried out by carbamylated Lys201 and Lys175, respectively. In the reactionmechanism proposed by Imker et al. (2007), Lys123 is the residue for de-protonation at C1.

1550 Ashidaet al.


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in a lack of enolization activity (Imker et al., 2007).

Therefore, Imker et al. proposed that a candidate residueto abstract the proton is Lys123, while the carbamylated

Lys201 is involved in de-protonation in photosynthetic

RuBisCO (Fig. 6). Interestingly, Lys123 is conserved in

RLPs of B. subtilis, M. aeruginosa, and G. kaustophilus,which all show DK-MTP-1-P enolase activity (Fig. 2),

suggesting that Lys123 is the essential residue for DK-MTP-1-P enolase activity in all RLPs. Considering

conservation of Lys123, Lys175, and Lys201 in DK-

MTP-1-P enolase activity, all three residues should

contribute to the enolization of DK-MTP-1-P. However,

it is unclear whether Lys175 and Lys201 are involved in

re- and de-protonation, as they are in photosynthetic

RuBisCO (Fig. 6). If Lys123 played a role in the

abstraction of protons in DK-MTP-1-P enolase, in spite

of the fact that Lys201 was carbamylated, why was

Lys201 carbamylated in RLP, as it is in photosynthetic

RuBisCO? It was reported by Imkar et al. (2007) thatthe carbamylation at Lys201 increased the kcat/Km for the

enolase reaction of G. kaustophilus RLP. However, therole of carbamylation on Lys201 is still unknown in

RLP. Further analysis will reveal the functional relation-

ship of carbamylation on Lys201 between RLPs and

photosynthetic RuBisCO. Three RLPs also had conserved

residues at Gly401 and Gly403, relative to the P1

phosphate-binding motif of photosynthetic RuBisCO

(Fig. 2) (Hartman and Harpel, 1994). In G. kaustophilusRLP, the P1 phosphate oxygens of DK-H-1-P form

hydrogen bonds to amide groups of the backbone of

Gly401 and Gly403 (Imker et al., 2007). DK-MTP-1-Penolase and photosynthetic RuBisCO utilize the common

conserved P1-binding motif to bind the substrate P1

phosphate group.

Are all RLPs acting as the DK-MTP-1-P enolases?

Many bacteria possess genes for RLPs, as shown in the

phylogenetic tree (Fig. 1). Are all RLPs acting as DK-

MTP-1-P enolases, as is the case in B. subtilis,

M. aeruginosa, and G. kaustophilus?As described above, RLPs from B. subtilis, M. aeruginosa,

and G. kaustophilus have conserved residues Lys123,Lys175, Lys201, Asp203, and Glu204 that are essential

for the enolization reaction of DK-MTP-1-P enolase

(Fig. 2). Other RLPs from group a together with those ofBacillus species and of cyanobacteria also have conservedresidues Lys123, Lys175, Lys201, Asp203, and Glu204

(Fig. 2). In addition, the genes for RLPs from B.licheniformis, B. cereus, and B. anthracis, and from other

Bacillus species are members of the mtnWXBD operon. Thegene for RLP from the cyanobacterium Lyngbya sp. PCC8106 is also predicted to form this operon with homologous

genes for mtnB and mtnE. These observations suggest that

these RLPs in group a also function as the DK-MTP-1-Penolase in the methionine salvage pathway. However, the

RLPs of R. rubrum and R. palustris, in the same group a,have conserved residues Lys175, Lys201, and Asp203, but

Lys123 and Glu204 are replaced with asparagine and

histidine, respectively (Fig. 2). Interestingly, it was reported

that R. rubrum and R. palustris utilize the methionine

salvage pathway because they can grow using MTA as thesole source of sulphur (Tabita et al., 2007). This resultimplies that R. rubrum and R. palustris RLPs function asthe DK-MTP-1-P enolase in these bacteria. Considering the

result of mutational analysis at Lys123 in G. kaustophilusRLP, these RLPs may not be the DK-MTP-1-P enolase,

because they do not have conserved Lys123. In R. rubrumand R. palustris, the reaction step of DK-MTP-1-P enolasemay be catalysed by a photosynthetic form II RuBisCO and

not by RLPs, because R. rubrum RuBisCO can catalyse thisreaction, although at a very low rate (described below)

(Ashida et al., 2003). As in the case of R. rubrum and

R. palustris, group a seem to include RLPs with other

functions, distinct from DK-MTP-1-P enolase. Tabita et al.(2007) reported a phylogenetic tree of RLPs produced using

a large number of RLP sequences. In Tabitas phylogenetic

tree, group a was further classified into two subgroups,

RLPs for DK-MTP-1-P enolase and photosynthetic bacterial

RLPs. If the phylogenetic tree is examined in detail, RLPs

of group a can be divided into two subgroups, a1 including

B. subtilis DK-MTP-1-P enolase and a2 including RLPsfrom photosynthetic bacteria, in agreement with Tabitas

classification. This classification of RLPs is in accord with

the prediction of function. RLP is the DK-MTP-1-P enolase,

based on conservation of predicted essential residues,

and, therefore, it is predicted that RLPs in group a1 are

DK-MTP-1-P enolases, but group a2 RLPs are not.However, Lys201 is replaced by glutamine in B. clausiiRLP of group a1 (Fig. 2) and this micro-organism does

not possess homologous genes for other enzymes

functioning in the methionine salvage pathway, in spite

of B. clausii being a Bacillus species. Likewise,

A. fulgidus RLP in group a1 is exceptional in thatLys123 is not conserved (Fig. 2) and, in addition, the

homologous genes encoding other enzymes for

methionine salvage cannot be found. These facts

suggest that these RLPs may not function as DK-MTP-1-P

enolase and that some RLPs classified in group a1

possess a different function. On the other hand, in RLPs in

groups b and c, Lys123 is replaced by asparagine, and,therefore, these RLP groups should not function as DK-

MTP-1-P enolase.

The function of RLPs from species other than Bacillusspecies and cyanobacteria is unclear. Hanson and Tabita

(2001) reported that C. tepidum RLP, included in RLPgroup c, is involved in sulphur oxidation and oxidativestress, suggesting that this RLP functions in other

biological process.



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Evolutionary and functional linkage between RLPand photosynthetic RuBisCO

Evolutionary and functional linkage between RLP and

photosynthetic RuBisCO has attracted interest. It is con-

cluded that there is functional linkage of RLP and RuBisCO

in the DK-MTP-1-P enolase reaction, because the catalytic

reaction and the substrate of DK-MTP-1-P enolase resemble

those of RuBisCO. Surprisingly, introduction of the gene forRuBisCO from the photosynthetic bacterium R. rubrumrescued the growth of an RLP-deficient B. subtilis mutant

on medium containing MTA as the sole sulphur source (Fig.

5d). In addition, recombinant R. rubrum RuBisCO showed

a very low, but significant level of DK-MTP-1-P activity in

vitro (Ashida et al., 2003). This suggested that photosyn-

thetic RuBisCO could catalyse the DK-MTP-1-P enolase

reaction, and that there is a functional link between RLP and

RuBisCO. It is therefore speculated that RLP and photosyn-

thetic RuBisCO evolved from the same ancestor protein.

Gupta proposed a hypothesis for linear evolution of

bacteria by indel analysis using highly conserved domains

of proteins that all bacteria commonly possess (Gupta,

1998; Gupta et al., 1999). In this hypothesis, low G+C

Gram-positive bacteria, including Bacillus species, would

have features of the most ancient bacteria, rather than

Archaea and Bacteria possessing RLPs and RuBisCO. If

the molecular evolution of RLP and photosynthetic

RuBisCO is discussed following Guptas hypothesis, RLP

in the methionine salvage pathway might have evolved

earlier than photosynthetic RuBisCO. Furthermore, photo-

synthetic RuBisCO might have evolved from an ancestral

DK-MTP-1-P enolase (Ashida et al., 2005).If this hypothesis is correct, the methionine salvage

pathway would be one of the most ancient metabolicpathways. However, ancient organisms might not have

utilized this pathway to recycle the reduced sulphur source,

because the concentration of hydrogen sulphide would have

been high in the environments where the most ancient

organisms initially emerged (Delano, 2001). It is therefore

thought that the methionine salvage pathway was not part of

ancient metabolism and that a catalytically distinct ancestor

protein of RLPs, functioning in the methionine salvage

pathway, might have already existed. Interestingly, the

methionine-salvaging enzymes, MtnK, A, B, and W, from

B. subtilis can catalyse sequential reactions for ribose,

in addition to the substrate, MTR (Imker et al., 2007).

Ribose is converted to 2-hydroxy-3-keto-5-hydroxypent-1-ene 1-phosphate by sequential reactions catalysed by four

methionine-salvaging enzymes including RLP (Imker et al.,

2007). It has been predicted that ribose was formed in the

chemical evolution era and was an essential compound in the

RNA world (Joyce, 2002). Ribose is one of the most ancient

compounds on earth, older than MTR. Considering that

methionine-salvaging enzymes can utilize ribose, an ances-

tral metabolic pathway of the methionine salvage pathway

might have been involved in some form of ribose metabo-

lism, incorporating the sequential reactions catalysed by themethionine-salvaging enzymes. In other words, an ancestral

RLP protein might be an enolase enzyme that functioned in

ancient ribose metabolism.

Concluding remarks

Many genome projects have revealed that Bacteria and

Archaea possess genes for RLPs that cannot catalyse either

the carboxylase or the oxygenase reactions of photosyn-

thetic RuBisCO. These facts provide the fourth form of

RuBisCO enzymes. The present studies have revealed that

one member of the fourth form, B. subtilis RLP, is theenolase enzyme functioning in the methionine salvage

pathway. In addition, RLPs from the cyanobacterium,

M. aeruginosa, and G. kaustophilis catalyse the enolasereaction in methionine salvage. Comparing the steps in the

catalytic cycle between these RLPs and RuBisCO, it was

found that RLPs catalysed a very similar reaction to

photosynthetic RuBisCO, for a substrate with a similarstructure to RuBP. In addition, both enzymes utilized the

same amino acid residues to catalyse each reaction,

suggesting that RLP and photosynthetic RuBisCO might

have evolved from the same ancestral protein. Further-

more, considering the molecular evolution of RuBisCObased on Guptas evolutionary hypothesis of micro-

organisms, the hypothesis is proposed that RLP, function-

ing as an enolase, is the ancestral protein of RuBisCO.

Thus, studies on RLPs have suggested an answer to the

fundamental question What is the origin of photosynthetic

RuBisCO? However, there remains limited information

about RLPs and, therefore, further analysis of evolutionary

and functional relationships between RLPs and RuBisCOis needed to answer to this question fully. In particular,

functional characterization of many RLPs is required to

establish the molecular evolution of RuBisCO.

Research on RLPs should provide useful information, not

only for evolutionary studies, but also to improve RuBisCO

efficiency. CO2 fixation by RuBisCO is the limiting step in

plant photosynthesis. Hence, inefficient RuBisCO is the

obvious target to improve efficiency of photosynthesis in

plants. To improve RuBisCO to Super-RuBisCO with

high CO2 specificity, depressed reactivity for O2, and

a high CO2-fixing catalytic rate, it is necessary to know the

amino acid residues involved in catalysis of RuBisCO.

Analysis of the functional and structural relationships

between RLPs and photosynthetic RuBisCO should pro-

vide important information on the structure and amino acid

residues involved in carboxylase or oxygenase reactions.

Acknowledgements

The authors thank Drs Naotake Ogasawara and Kazuo Kobayashifor help and discussion in molecular manipulation of Bacillus. We

1552 Ashidaet al.


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are grateful to Dr Chojiro Kojima for 1H-NMR analysis. This workwas supported partly by a Grant in Aid (17208031 and 18688021)for Scientific Research from the Japan Society for the Promotion ofScience (JSPS), partly by a grant (FY2004-2006) for GeneralScience and Technology from the Asahi Glass Foundation, andpartly by a grant (FY2005-2007) from the Nissan ScienceFoundation.

References

Andrews TJ, Lorimer GH. 1987. Rubisco: structure, mechanisms,and prospects for improvement. In: Hatch M, Boardman N, eds.The biochemistry of plants, Vol. 10. New York: Academic Press,131218.

Andrews TJ, Whitney SM. 2003. Manipulating ribulose bisphos-phate carboxylase/oxygenase in the chloroplasts of higher plants.Archives of Biochemistry and Biophysics 414, 159169.

Ashida H, Danchin A, Yokota A. 2005. Was photosyntheticRuBisCO recruited by acquisitive evolution from RuBisCO-likeproteins involved in sulfur metabolism? Research in Microbiol-ogy 156, 611618.

Ashida H, Saito Y, Kojima C, Kobayashi K, Ogasawara N,

Yokota A. 2003. A functional link between RuBisCO-like proteinof Bacillus and photosynthetic RuBisCO. Science 302, 286290.Ashida H, Saito Y, Kojima C, Yokota A. 2007. Enzymatic

characterization of 5-methylthioribulose-1-phosphate dehydrataseof the methionine salvage pathway from Bacillus subtilis.Bioscience, Biotechnology and Biochemistry 71, 20212028.

Balakrishnan R, Frohlich M, Rahaim PT, Backman K,Yocum RR. 1993. Appendix. Cloning and sequence of the geneencoding enzyme E-1 from the methionine salvage pathway ofKlebsiella oxytoca. Journal of Biological Chemistry 268, 2479224795.

Berger BJ, English S, Chan G, Knodel MH. 2003. Methionineregeneration and aminotransferases in Bacillus subtilis, Bacilluscereus, and Bacillus anthracis. Journal of Bacteriology 185,24182431.

Burstenbinder K, Rzewuski G, Wirtz M, Hell R, Sauter M.2007. The role of methionine recycling for ethylene synthesis inArabidopsis. The Plant Journal 49, 238249.

Carre-Mlouka A, Mejean A, Quillardet P, et al. 2006. A newRubisco-like protein coexists with a photosynthetic Rubisco inthe planktonic cyanobacteria Microcystis. Journal of BiologicalChemistry 281, 2446224471.

Cleland WW, Andrews TJ, Gutteridge S, Hartman FC,Lorimer GH. 1998. Mechanism of Rubisco: the carbamate asgeneral base. Chemical Reviews 98, 549562.

Delano JW. 2001. Redox history of the Earths interior sinceapproximately 3900 Ma: implications for prebiotic molecules.Origins of Life and Evolution of the Biosphere 31, 311341.

Ellis RJ. 1979. Most abundant protein in the world. Trends inBiochemical Sciences 4, 241244.

Estelle M, Hanks J, McIntosh L, Somerville C. 1985. Site-

specific mutagenesis of ribulose-1,5-bisphosphate carboxylase/oxygenase. Evidence that carbamate formation at Lys191 isrequired for catalytic activity. Journal of Biological Chemistry260, 95239526.

Furfine ES, Abeles RH. 1988. Intermediates in the conversion of5#-S-methylthioadenosine to methionine in Klebsiella pneumo-niae. Journal of Biological Chemistry 263, 95989606.

Garcia-Castellano JM, Villanueva A, Healey JH, Sowers R,Cordon-Cardo C, Huvos A, Bertino JR, Meyers P, Gorlick R.2002. Methylthioadenosine phosphorylase gene deletions arecommon in osteosarcoma. Clinical Cancer Research 8, 782787.

Grundy F, Henkin T. 2002. Synthesis of serine, glycine, cysteine,and methionine. In: Sonenshein A, Hoch J, Losick R, eds.

Bacillus subtilis and its closest relatives: from genes to cells.Washington, DC: American Society for Microbiology, 245254.

Gupta RS. 1998. Protein phylogenies and signature sequences:a reappraisal of evolutionary relationships among archaebacteria,

eubacteria, and eukaryotes. Microbiology and Molecular BiologyReviews 62, 14351491.

Gupta RS, Mukhtar T, Singh B. 1999. Evolutionary relationships

among photosynthetic prokaryotes (Heliobacterium chlorum,Chloroflexus aurantiacus, cyanobacteria, Chlorobium tepidumand proteobacteria): implications regarding the origin of photo-

synthesis. Molecular Microbiology 32, 893906.Gutteridge S, Lorimer G, Pierce J. 1988. Details of the reactions

catalyzed by mutant forms of Rubisco. Plant Physiology andBiochemistry 26, 675682.

Hanson TE, Tabita FR. 2001. A ribulose-1,5-bisphosphatecarboxylase/oxygenase (RubisCO)-like protein from Chlorobiumtepidum that is involved with sulfur metabolism and the responseto oxidative stress. Proceedings of the National Academy ofSciences, USA 98, 43974402.

Hartman FC, Harpel MR. 1994. Structure, function, regulation,and assembly ofD-ribulose-1,5-bisphosphate carboxylase/oxygen-

ase. Annual Review of Biochemistry 63, 197234.

Hartman FC, Soper TS, Niyogi SK, Mural RJ, Foote RS,Mitra S, Lee EH, Machanoff R, Larimer FW. 1987. Functionof Lys-166 of Rhodospirillum rubrum ribulosebisphosphatecarboxylase/oxygenase as examined by site-directed mutagenesis.

Journal of Biological Chemistry 262, 34963501.Hudson GS, Evans JR, von Caemmerer S, Arvidsson YB,

Andrews TJ. 1992. Reduction of ribulose-1,5-bisphosphatecarboxylase/oxygenase content by antisense RNA reduces photo-

synthesis in transgenic tobacco plants. Plant Physiology andBiochemistry 98, 294302.

Imker HJ, Fedorov AA, Fedorov EV, Almo SC, Gerlt JA. 2007.Mechanistic diversity in the RuBisCO superfamily: the enolase

in the methionine salvage pathway in Geobacillus kaustophilus.Biochemistry 46, 40774089.

Joyce GF. 2002. The antiquity of RNA-based evolution. Nature

418, 214221.Ma JF, Shinada T, Matsuda C, Nomoto K. 1995. Biosyn-

thesis of phytosiderophores, mugineic acids, associated withmethionine cycling. Journal of Biological Chemistry 270,1654916554.

Marchitto KS, Ferro AJ. 1985. The metabolism of 5#-methyl-thioadenosine and 5-methylthioribose 1-phosphate in Saccharo-myces cerevisiae. Journal of General Microbiology 131, 21532164.

Mauser H, King WA, Gready JE, Andrews TJ. 2001. CO(2)fixation by Rubisco: computational dissection of the key steps of

carboxylation, hydration, and CC bond cleavage. Journal of theAmerican Chemical Society 123, 1082110829.

Montange RK, Batey RT. 2006. Structure of the S-adenosylme-thionine riboswitch regulatory mRNA element. Nature 441,

11721175.Murphy BA, Grundy FJ, Henkin TM. 2002. Prediction of gene

function in methylthioadenosine recycling from regulatory sig-

nals. Journal of Bacteriology 184, 23142318.Myers RW, Wray JW, Fish S, Abeles RH. 1993. Purification and

characterization of an enzyme involved in oxidative carbon

carbon bond cleavage reactions in the methionine salvage

pathway of Klebsiella pneumoniae. Journal of Biological Chem-istry 268, 2478524791.

Pearce FG, Andrews TJ. 2003. The relationship betweenside reactions and slow inhibition of ribulose-bisphosphate



12/12

carboxylase revealed by a loop 6 mutant of the tobacco enzyme.Journal of Biological Chemistry 278, 3252632536.

Roy H, Andrews TJ. 2000. Rubisco: assembly and mechanism. In:Leegood R, Sharkey T, von Caemmerer S, eds. Photosynthesis:physiology and metabolism, Vol. 9. Springer: Dordrecht, TheNetherlands, 5383.

Saito Y, Ashida H, Kojima C, Tamura H, Matsumura H, Kai Y,Yokota A. 2007. Enzymatic characterization of 5-methylthio-ribose 1-phosphate isomerase from Bacillus subtilis. Bioscience,

Biotechnology and Biochemistry 71, 20212028.Sekowska A, Danchin A. 2002. The methionine salvage pathway

in Bacillus subtilis. BMC Microbiology 2, 8.Sekowska A, Denervaud V, Ashida H, Michoud K, Haas D,

Yokota A, Danchin A. 2004. Bacterial variations on themethionine salvage pathway. BMC Microbiology 4, 9.

Sekowska A, Kung HF, Danchin A. 2000. Sulfur metabolism inEscherichia coli and related bacteria: facts and fiction. Journal ofMolecular Microbiology and Biotechnology 2, 145177.

Sekowska A, Mulard L, Krogh S, Tse JK, Danchin A. 2001.MtnK, methylthioribose kinase, is a starvation-induced protein inBacillus subtilis. BMC Microbiology 1, 15.

Tabita FR. 1999. Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: a different perspective. Photosynthesis Research 60, 128.

Tabita FR, Hanson TE, Li H, Satagopan S, Singh J, Chan S.2007. Function, structure, and evolution of the RubisCO-likeproteins and their RubisCO homologs. Microbiology and Molec-ular Biology Reviews 71, 576599.

von Caemmerer S, Millgate A, Farquhar GD, Furbank RT.1997. Reduction of ribulose-1,5-bisphosphate carboxylase/oxy-genase by antisense RNA in the C4 plant Flaveria bidentis leads

to reduced assimilation rates and increased carbon isotopediscrimination. Plant Physiology 113, 469477.

Winkler WC, Nahvi A, Sudarsan N, Barrick JE, Breaker RR.2003. An mRNA structure that controls gene expression by bindingS-adenosylmethionine. Nature Structural Biology 10, 701707.

Wray JW, Abeles RH. 1993. A bacterial enzyme that catalyzesformation of carbon monoxide. Journal of Biological Chemistry268, 2146621469.

Wray JW, Abeles RH. 1995. The methionine salvage pathway inKlebsiella pneumoniae and rat liver. Identification and character-ization of two novel dioxygenases. Journal of BiologicalChemistry 270, 31473153.

1554 Ashidaet al.

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