microbial enzymes involved in carbon dioxide fixation

9
JOURNAL OFBIOSCIENCE AND BIOENGINEEFUNG Vol.94, No. 6,497-505.2002 REVIEW Microbial Enzymes Involved in Carbon Dioxide Fixation HARUYUKI ATOMI’ Department of Synthetic Chemishy and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan’ Received9 September 2002/Accepted 17 September 2002 This review focuses on the enzymes involved in two microbial carbon dioxide fixation path- ways, the Calvin-Benson-Bassham cycle and the reductive tricarboxylic acid cycle. The function, structural features, and gene regulation of microbial ribulose 1,5-bisphosphate carboxylase/oxy- genase (Rubisco), a key enzyme of the Calvin-Benson-Bassham cycle, is described. Some recent findings on Rubisco from archaea and Rubisco-like proteins are also outlined. In the final section, biochemical features of the key enzymes in the reductive tricarboxylic acid cycle are reviewed. [Key words: carbon dioxide fixation, ribulose 1,5_bisphosphate carboxylase/oxygenase, Rubisco, Calvin-Benson- Bassham cycle, ATP-citrate lyase, reductive tricarboxylic acid cycle] Heterotrophic organisms, including ourselves, other ani- mals, and a huge variety of microorganisms, continuously consume organic carbon compounds in order to maintain life. These compounds are used as carbon and energy sources, and are eventually oxidized to the form of carbon dioxide. The organic carbon on our planet is replenished in a biosynthetic manner owing to the activities of autotrophic organisms. Autotrophic organisms utilize the energy ob- tained from light or inorganic compounds to reduce carbon dioxide and synthesize organic material. Although plants and algae are known to be solely dependent on the Calvin- Benson-Bassham cycle (Calvin cycle) to fix carbon di- oxide, four pathways are known to function in autotrophic prokaryotes. These are: (i) the Calvin cycle (l), (ii) the acetyl-CoA pathway (2, 3), (iii) the 3-hydroxypropionate cycle (4), and (iv) the reductive tricarboxylic acid cycle (5, 6). This review briefly introduces the enzymes involved in the Calvin cycle and the reductive tricarboxylic acid cycle, and also describes the enzymatic features of a recently iden- tified ribulose 1,5-bisphosphate carboxylaseloxygenase from archaea. Although not dealt with here, there has been a tre- mendous advance in our understanding of the 3-hydroxy- propionate cycle in recent months, which is reported in Refs. 4, 7, and 8. I. THE CALVIN-BENSON-BASSHAM CYCLE The Calvin cycle is by far the most well-known and stud- ied CO, fixation pathway. There is only one enzyme in the cycle that fixes carbon dioxide, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) (9, 10). Rubisco is con- sidered to be the most abundant enzyme on our planet and is e-mail: [email protected] phone: +81-(0)75-753-5572 fax: +81-(0)75-753-4831 therefore recognized as the major gateway through which inorganic carbon (CO,) enters the biosphere. Besides the photosynthetic eukaryotic organisms, numerous prokaryotes have also been found to rely on the Calvin cycle for CO, fixation, and many more have been shown to at least harbor a Rubisco (11). It seems that the Calvin cycle is utilized by purple nonsulfur bacteria (Rhodobacter, Rhodospirillum, Rhodopseudomonas) and purple sulfur bacteria (Chro- matium), cyanobacteria (Synechococcus, Anacystis, Ana- baena), along with hydrogen bacteria (Ralstonia, Hydro- genovibrio) and other chemoautotrophs (Thiobacillus). The Calvin cycle consists of 13 enzymatic reactions (Fig. I), among which the Rubisco-catalyzed reaction is the only one that fixes CO,: one molecule of ribulose 1,5-bisphos- phate (RuBP), CO,, and H,O are converted into two mole- cules of 3-phosphoglycerate (3-PGA). The role of the other 12 reactions is to regenerate RuBP. In total, after 3 mole- cules of CO, are fixed to 3 molecules of RuBP, 6 molecules of 3-PGA are formed. Five of these are used to regenerate 3 molecules of RuBP. The remaining single molecule of 3- PGA is used for the biosynthesis of all cell material. Three enzymes can be considered unique to the Calvin cycle: Rubisco, phosphoribulokinase (PRK), and sedoheptulose bisphosphatase (SBPase). The activities of the other en- zymes are shared with the gluconeogenesis pathway and the pentose phosphate cycle (1). II. RUBISCO As noted above, Rubisco proteins from various sources have been identified and examined. Besides the recently identified Type III and Type IV enzymes (see below), Rubiscos can be structurally divided into two types (10). The Type I enzyme is the dominant Rubisco and is found in all plants, algae, cyanobacteria, and most chemoautotrophs. 497

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Page 1: Microbial Enzymes Involved in Carbon Dioxide Fixation

JOURNAL OF BIOSCIENCE AND BIOENGINEEFUNG Vol. 94, No. 6,497-505.2002

REVIEW

Microbial Enzymes Involved in Carbon Dioxide Fixation HARUYUKI ATOMI’

Department of Synthetic Chemishy and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan’

Received 9 September 2002/Accepted 17 September 2002

This review focuses on the enzymes involved in two microbial carbon dioxide fixation path- ways, the Calvin-Benson-Bassham cycle and the reductive tricarboxylic acid cycle. The function, structural features, and gene regulation of microbial ribulose 1,5-bisphosphate carboxylase/oxy- genase (Rubisco), a key enzyme of the Calvin-Benson-Bassham cycle, is described. Some recent findings on Rubisco from archaea and Rubisco-like proteins are also outlined. In the final section, biochemical features of the key enzymes in the reductive tricarboxylic acid cycle are reviewed.

[Key words: carbon dioxide fixation, ribulose 1,5_bisphosphate carboxylase/oxygenase, Rubisco, Calvin-Benson- Bassham cycle, ATP-citrate lyase, reductive tricarboxylic acid cycle]

Heterotrophic organisms, including ourselves, other ani- mals, and a huge variety of microorganisms, continuously consume organic carbon compounds in order to maintain life. These compounds are used as carbon and energy sources, and are eventually oxidized to the form of carbon dioxide. The organic carbon on our planet is replenished in a biosynthetic manner owing to the activities of autotrophic organisms. Autotrophic organisms utilize the energy ob- tained from light or inorganic compounds to reduce carbon dioxide and synthesize organic material. Although plants and algae are known to be solely dependent on the Calvin- Benson-Bassham cycle (Calvin cycle) to fix carbon di- oxide, four pathways are known to function in autotrophic prokaryotes. These are: (i) the Calvin cycle (l), (ii) the acetyl-CoA pathway (2, 3), (iii) the 3-hydroxypropionate cycle (4), and (iv) the reductive tricarboxylic acid cycle (5, 6). This review briefly introduces the enzymes involved in the Calvin cycle and the reductive tricarboxylic acid cycle, and also describes the enzymatic features of a recently iden- tified ribulose 1,5-bisphosphate carboxylaseloxygenase from archaea. Although not dealt with here, there has been a tre- mendous advance in our understanding of the 3-hydroxy- propionate cycle in recent months, which is reported in Refs. 4, 7, and 8.

I. THE CALVIN-BENSON-BASSHAM CYCLE

The Calvin cycle is by far the most well-known and stud- ied CO, fixation pathway. There is only one enzyme in the cycle that fixes carbon dioxide, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) (9, 10). Rubisco is con- sidered to be the most abundant enzyme on our planet and is

e-mail: [email protected] phone: +81-(0)75-753-5572 fax: +81-(0)75-753-4831

therefore recognized as the major gateway through which inorganic carbon (CO,) enters the biosphere. Besides the photosynthetic eukaryotic organisms, numerous prokaryotes have also been found to rely on the Calvin cycle for CO, fixation, and many more have been shown to at least harbor a Rubisco (11). It seems that the Calvin cycle is utilized by purple nonsulfur bacteria (Rhodobacter, Rhodospirillum, Rhodopseudomonas) and purple sulfur bacteria (Chro- matium), cyanobacteria (Synechococcus, Anacystis, Ana- baena), along with hydrogen bacteria (Ralstonia, Hydro- genovibrio) and other chemoautotrophs (Thiobacillus).

The Calvin cycle consists of 13 enzymatic reactions (Fig. I), among which the Rubisco-catalyzed reaction is the only one that fixes CO,: one molecule of ribulose 1,5-bisphos- phate (RuBP), CO,, and H,O are converted into two mole- cules of 3-phosphoglycerate (3-PGA). The role of the other 12 reactions is to regenerate RuBP. In total, after 3 mole- cules of CO, are fixed to 3 molecules of RuBP, 6 molecules of 3-PGA are formed. Five of these are used to regenerate 3 molecules of RuBP. The remaining single molecule of 3- PGA is used for the biosynthesis of all cell material. Three enzymes can be considered unique to the Calvin cycle: Rubisco, phosphoribulokinase (PRK), and sedoheptulose bisphosphatase (SBPase). The activities of the other en- zymes are shared with the gluconeogenesis pathway and the pentose phosphate cycle (1).

II. RUBISCO

As noted above, Rubisco proteins from various sources have been identified and examined. Besides the recently identified Type III and Type IV enzymes (see below), Rubiscos can be structurally divided into two types (10). The Type I enzyme is the dominant Rubisco and is found in all plants, algae, cyanobacteria, and most chemoautotrophs.

497

Page 2: Microbial Enzymes Involved in Carbon Dioxide Fixation

498 ATOM1 J. BIOSCI. BIOENG.,

COOH CH,O-@ Ribulose

CH,OH Ribose

HCOH

dH,O-@ Ru bisco

C=O B-phosphate C=O 5-phosphate HCOH kinase HCOH isomerasa

- HCOH - 3-Phosphoglycarate

CH,O-@

1,5-bisphosphate Ribulose Sphosphate

CHO

HCOH

HCOH

HCOH

CH,O-@

5-phosphate

4 Phosphoglycerate kinase

Fructose G-phosphate

1,3-Bisphcsphoglycerate

OH

Glyceraldehyde 3-phosphate

Dihydroxyacetone phosphate

Sedoheptulose 1,7-bisphosphate

FIG. 1. The Calvin-Benson-Bassham cycle.

The enzyme consists of two distinct subunits, a large (L) subunit (50-55 kDa), which is the catalytic subunit of the enzyme, and a small (S) subunit (12-18 kDa). Dimerization of two L subunits is necessary to generate two catalytic cen- ters per L, dimer, and the quatemary structure of a Type I enzyme is L,S,. In eukaryotic cells, the L subunit is encoded on the chloroplast genome, while the genes for the S subunit are located on the nuclear genome. In contrast to Type I, the Type II enzyme has been identified in only a relatively small number of bacteria. This enzyme is composed solely of L subunits and is usually found in an L, form. Rhodospirillum rubrum harbors a single Type II Rubisco (12), while various species of Rhodobacter (13), Thiobacillus (14), and Hydro- genovibrio (15) have been found to contain both Type I and Type II. An interesting exception in the distribution of the two types of Rubiscos is that the enzyme found in the chlo- roplasts of eukaryotic dinoflagellates is a Type II and is en- coded on the nuclear chromosome (16).

A wealth of information is available on the biochemical features of Rubiscos, and concise reviews have been pub- lished (9, 17). In general, Rubiscos are considered far from the ideal catalyst, with a turnover rate of 2-5 s-l; various side reactions have also been found to occur. Besides the carboxylase activity described above, all Rubiscos are known to display an additional oxygenase activity, in which an oxygen molecule, competing with CO, for the enzyme- bound eno-diolate of RuBP, reacts with RuBP to form 3-

PGA and phosphoglycolate. The latter product is subse- quently oxidatively metabolized via photorespiration, lead- ing to a net loss in carbon dioxide fixation. Although the relevance of this activity in vivo has yet to be determined, exhaustive research has been undertaken in attempts to understand and/or increase the discriminating ability of Rubisco towards CO,. This will hopefully lead to an in- crease in the net photosynthesis of plants (18, 19).

The ability of a particular Rubisco to discriminate be- tween carboxylation and oxygenation is an intrinsic prop- erty, often evaluated by determining its specificity factor, or r value. This is defined as Vco,&(Vo&,~, where Vcoz and Vol represent the maximal velocity for CO, and O,, respec- tively, and KcO, and kYo2 represent the respective Michaelis constants. Type I Rubiscos from higher plants such as spin- ach display a rvalue of approximately 80, while those from bacteria or green algae exhibit values between 40 and 60. The r values of Type II Rubiscos are even lower, ranging from approximately 10 to 20 (11). A number of Type I Rubiscos with relatively high r values have been identified from marine algae such as Porphyridium (z= 129) and Cylindrotheca (r=106-111) (20). The enzymes from the thermophilic red algae Galdieria and Cyanidium have r values of 238 and 222, respectively (21).

Extensive research has been performed on Rubiscos from diverse bacteria, including Rhodospirillum rubrum (22), Rhodobacter capsulatus (23), Rhodobacter sphaeroides

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VOL. 94.2002 MICROBIAL CO, FIXATION 499

(24), Ralstonia eutropha (Alcaligenes eutrophus) (25), Hy- drogenovibrio marinus (15), Thiobacillus denitrt$cans (14), Anacystis nidulans (26) and Synechococcus sp. PCC6301 (27). The crystal structures of the Type I enzyme from Synechococcus sp. PCC6301 (28) and R. eutropha (25), along with the Type II enzyme from R. rubrum (29), have been determined. Besides the biochemical and structural aspects of the enzymes, the regulation of Rubisco gene ex- pression has recently been drawing attention, particularly in bacteria harboring two or more Rubisco genes.

The purple nonsulfur bacterium R. capsulatus displays extreme versatility in carbon metabolism (30). Cells exhibit chemoheterotrophic growth on a variety of carbon sources under aerobic conditions, and can also grow chemoauto- trophically with hydrogen and CO,. In addition, the strain exhibits both photoheterotrophic and photoautotrophic growth. Interestingly, the Calvin cycle provides two distinct functions in R. capsulatus depending on the growth condi- tions. The cycle in autotrophically grown cells contributes to the fixing of CO,, in the usual manner. However, under photoheterotrophic conditions, R. capsulatus utilizes the Calvin cycle in order to release excess reducing equivalents to the preferred electron acceptor, CO, (31). R. capsulatus harbors two distinct types of Rubisco, the Type I enzyme encoded by the cbbL and cbbS genes in the ebb, operon, and the Type II enzyme encoded by the cbbM gene in the ebb,, operon (30). The Type I enzyme is the predominant Rubisco during photoautotrophic growth, and is not synthesized dur- ing photoheterotrophic growth. The Type II enzyme is pro- duced under all growth conditions and can be supposed to contribute in balancing the redox poise of the cell during photoheterotrophic growth. However, gene disruption stud- ies have indicated that the Type II enzyme alone is also capable of supporting photoautotrophic growth in R. cap- sulatus (30).

The CbbR activator, a LysR-type transcriptional activa- tor, has been identified in a number of autotrophic bacteria dependent on the Calvin cycle, including R. sphaeroides and R. capsulatus. In R. sphaeroides, a single cbbR gene product is known to positively regulate the expression of both ebb, and ebb,, operons (32). In the case of R. cap- sulatus, there is a cbbR gene upstream of each of the ebb, and ebb,, operons, designated cbbR, and cbbR,,, respectively. Gene disruption of cbbR,, was found to strongly reduce the expression levels of a reporter gene under the control of the ebb,, promoter, whereas no significant effect was observed in a cbbR, disruptant, suggesting that the CbbR,, activator is mainly responsible for the regulation of the ebb,, operon (30). Further studies have indicated the involvement of the RegA-RegB two-component system in the regulation of R. capsulatus ebb operons (33). Reductions in the expression levels of both ebb operons were found in regA and regB mutant strains, and gene regulation dependent on growth conditions could no longer be observed. These findings, along with others (34), indicate the presence of an ex- tremely complex system in the regulation of ebb operons in purple nonsulfur bacteria.

III. ARCHAEAL RUBISCO

Recent studies have indicated that although a functional Calvin cycle has not yet been identified in archaea, some hyperthermophilic strains harbor proteins that exhibit Rubisco activity. These are Thermococcus kodakaraensis KODI (35-39), Methanococcus jannaschii (40), and Ar- chaeoglobus fulgidus (40), whose genome sequences have been published. 7: kodakaraensis KODl is a hyperthermo- philic archaeon isolated from a solfatara on Kodakara Is- land, Japan (41). The strain grows at temperatures between 65°C and lOO”C, is strictly anaerobic, and displays hetero- trophic growth on a variety of substrates, including amino acids, starch, oligosaccharides, and pyruvate. As enzymes from hyperthermophiles commonly exhibit extreme thermo- tolerance, they are expected to provide valuable information in terms of the structural factors that lead to protein stability. The enzymes are also a focus of attention in the field of biotechnology, where there is a constant demand for stable biocatalysts. A number of genes that encode enzymes with unique features have been identified from strain KODl, in particular DNA polymerase (42), DNA ligase (43,44), and chaperonins A and B (45) along with a variety of sugar- modifying enzymes (4649).

An open reading frame displaying -50% similarity to previously reported L subunits of Type I and Type II Rubiscos was identified on the genome of 7: kodakaraensis KODl (35). Similar open reading frames have also been found on the genomes of M. jannaschii and A. fulgidus, suggesting some kind of physiological role for these genes in hyperthermophilic archaea. Phylogenetic analysis of Rubisco sequences indicates that the archaeal Rubiscos, although diverse, cluster together in a branch distinct from those of previously identified Type I and Type II enzymes (Fig. 2). The putative Rubisco gene from T. kodakaraensis was expressed in Escherichia coli and the recombinant en- zyme (Tk-Rubisco) was analyzed. Activity measurements revealed that Tk-Rubisco was capable of catalyzing the car- boxylation and oxygenation of ribulose 1,5_bisphosphate, indicating the protein to be a bona$de Rubisco. In fact, as Tk-Rubisco displayed activity at temperatures as high as lOO”C, its specific activity is higher than any previously characterized Rubisco. Tk-Rubisco also exhibited an un- precedentedly high carboxylase specificity (a r value of ap- proximately 3 10 at 9O’C). As the primary and quaternary (see below) structures of Tk-Rubisco were distinct from those of previously characterized Rubiscos, the enzyme was designated a member of a novel Type III Rubisco family

(37). Northern and Western blot analyses indicated that the Tk-

Rubisco gene was transcribed and translated, and the pro- tein product was present in 7: kodakaraensis KODl cells. The use of polyclonal antibodies against the recombinant enzyme also indicated that the native enzyme in KODl cells is composed solely of large subunits, as in the case of the Type II Rubisco. The molecular mass of the native enzyme was estimated to be -450 kDa, while the calculated molecu- lar mass of the subunit was -50 kDa. Taking account of the fact that the minimal active unit of a Rubisco is a dimer, Tk- Rubisco was supposed to be an octamer or decamer. When

Page 4: Microbial Enzymes Involved in Carbon Dioxide Fixation

500 ATOMI

TYPE I

T. kodakaraensis

T.denihilicans-2

TYPE II \ M.jannaschii

8. sub tilis

0.1 -

A. fUgi&-1

\ C.limiwla

FIG. 2. Phylogenetic tree of representative Rubiscos from various sources. A. fulgidus-I, -2, rbcL1 and rbcL2 gene products from Ar- chaeoglobus fulgidus; B. subtilis, Bacillus subtilis; C. limicola, Chlo- robium limicola f. sp. thiosulfatophilum strain Tassajara; C. vinosum, Chromatium vinosum; Cylindrotheca, Cylindrotheca sp. strain Nl ; H. marinus-2, Type II enzyme from Hydrogenovibrio marinus; M jann- aschii, Methanococcus jannaschii; P. horikoshii, Pyrococcus horiko- shii; R. capsulatus-I, -2, Type I and Type II enzymes from Rhodo- batter capsulatus; R. eutropha, Ralstonia eutropha; R. rubrum, Rho- dospirillum rubrum; R. sphaeroides-I, -2, Type I and Type II enzymes from Rhodobacter sphoeroides; Synechococcus, Synechococcus sp. PCC6301; 7: denitrifcans-I, -2, Type I and Type II enzymes from Thiobacillus denitrifcons; I: Kodakaraensis, Thermococcus kodokaroensis KOD 1.

the purified enzyme was examined by electron microscopy, pentagonal images were clearly observed. These findings indicated that Tk-Rubisco is a decamer consisting only of

.I. Brom. BIOENG.,

large subunits, with a pentagonal ring-like structure (36). 2%Rubisco was crystallized, and the crystal structure was

determined at 2.8 A resolution (Fig. 3). Under electron mi- croscopical observation, 7%Rubisco displayed a novel (L,), decameric structure (38). Compared to previously known Type I enzymes, each L, dimer was inclined approximately 16’ to form a toroid-shaped decamer with unique L,-L, in- terfaces. Despite the low sequence homology, the monomer (L) and dimer (LJ structures of 7%Rubisco were well con- served compared with those of Type I and Type II enzymes. In its unique L,-L, interface, which includes the rims of the L-L dimerization surface, an intensive ionic network was observed. In particular, three neighboring residues (Glu63, Arg66, and Asp69) were considered likely to participate in these ionic interactions. Three single-mutant proteins (E63S, R66S, and D69S) and one triple-mutant protein (E63S/R66S/ D69S) were constructed by replacing the charged residues with serine. The wild-type (WT) and D69S proteins were decameric at all temperatures examined between 30°C and 90°C. The majority of E63S and R66S were decamers at 30°C but were found to gradually disassemble as the tem- perature was elevated (Fig. 4). E63S/R66S/D69S was ob- served in a dimeric form at all the temperatures examined. An interesting correlation was found between the subunit assembly and thermotolerance of the proteins. The denatur- ation temperature of the dimeric enzymes (E63S, R66S, E63YR66SiD69S) was approximately 95°C while those of the enzymes retaining a decameric structure (WT, D69S) were approximately 110°C (Fig. 5). Disassembly into tet- ramer or dimer units did not alter the slopes of the Arrhe- nius plots, indicating that the decameric structure had no effect on catalytic performance per se. The results indicated that the decameric assembly of 7%Rubisco contributes to enhancing the thermotolerance of the enzyme (39). Taking into account the growth temperature range of strain KODl

FIG 3. Crystal structure of Type III Rubisco from 7: Rodakaraensis KOD 1. A top view of the enzyme is shown on the left, and a side view on the right. Individual subunits (A-J) are displayed in different colors.

Page 5: Microbial Enzymes Involved in Carbon Dioxide Fixation

VOL. 94,2002 MICROBIAL CO, FIXATION 501

A BC A 1Omer B 4mer C 2mer

E63S A BC

: :

R66S D69S A ?!: A BC .,

FIG. 4. Subunit assembly of Tk-Rubisco and its mutant proteins at various temperatures. The left panel shows the elution profiles of gel perme- ation chromatography with purified Tk-Rubisco and its mutant proteins at 30°C. The other panels display the elution profiles of gel permeation chromatography with purified E63S, R66S, and D69S mutant proteins at various temperatures. All proteins were applied at an equivalent concen- tration of 1 mg/ml. The dotted lines A, B, and C represent retention times corresponding to decameric, tetrameric, and dimeric proteins.

(65-IOO‘C), the decameric structure of Tk-Rubisco can be considered essential for the stable presence of the enzyme in host cells.

In spite of the knowledge accumulated on the biochemi- cal and structural features of Tk-Rubisco, little progress has been made in elucidating the physiological role of the en- zyme. For it to function as one of the key enzymes of the Calvin cycle, other enzymes should also be present in the cells. Enzymes such as fructose 1,6-bisphosphatase (50) and fructose 1,6_bisphosphate aldolase (5 1) have been identified in 7: kodakaraensis. However, we have been unable to de-

I I I 1 i i i i 1 40 50 60 70 &I 90 loo 110 120

Temperature (“c)

FIG. 5. Differential scanning calorimetry of wild-type T/c-Rubisco and its mutant proteins E63S, R66S, D69S, and E63S/R66S/D69S.

tect activity of another key enzyme of the cycle, ribulose 5- phosphate kinase. Complete genome sequences from other archaeal strains also show no evidence of ribulose 5-phos- phate kinase orthologues on their chromosomes. Therefore, although Tk-Rubisco harbors Rubisco activity, it is likely that the protein has another enzyme activity or function that is necessary for the cells.

Recently, a growing number of microbial genome se- quences have become available, revealing the presence of additional ORFs displaying similarity to Rubiscos. Interest- ingly, some of these genes encode proteins that lack some key residues necessary for Rubisco activity as we know it, and are referred to as “Rubisco-like” proteins, or Type IV Rubiscos. These include genes from Bacillus subtilis, A. fulgidus, and the green sulfur bacteria Chlorobium tepidum and C. limicola. Valuable information has been provided through disruption experiments of the Type IV gene from C. tepidum (52). The resulting mutant displayed defects in sul- fur metabolism,’ along with a substantial accumulation of oxidative stress proteins, indicating that the protein might be involved in oxidative stress responses and/or sulfur me- tabolism. It may well be that these Type IV proteins, along with the archaeal Type III Rubiscos, commonly contribute to these pathways via a novel catalytic activity distinct from the activity necessary in the Calvin cycle.

IV. THE REDUCTIVE TRICARBOXYLIC ACID CYCLE

The green sulfur bacteria, including Chlorobium species, are known to utilize the reductive tricarboxylic cycle (RTCA cycle) in order to fix the CO, necessary for photo- autotrophic growth. The cycle was first proposed in 1966 by Evans et al., supported by the discovery of ferredoxin-de- pendent pyruvate synthase and 2-oxoglutarate synthase (5). Later, the presence of an ATP-dependent citrate lyase was

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502 ATOM1 J. BIOSCI. B~OENF..

also confirmed, thus allowing a reverse flux of the well- known tricarboxylic cycle (53). In the RTCA cycle, one molecule of acetyl-CoA is synthesized from two molecules of CO,. The acetyl-CoA can be converted to pyruvate and phosphoenolpyruvate, which can then be used to regenerate the intermediates of the cycle in an anaplerotic manner.

Recent studies have been carried out on RTCA cycle enzymes in various bacteria, including Desulfobacter hy- drogenophilus, Hydrogenobacter thermophilus, Chlorobium tepidum, and Chlorobium limicola. The cycle has also been investigated in the archaeon, Thermoproteus neutrophilus. In the strictly anaerobic chemoautotroph D. hydrogenophi- lus, all enzymes of the RTCA cycle were confirmed to be present at specific activity levels that would be sufficient for autotrophic growth. Additionally, key enzyme activities of the Calvin cycle and acetyl-CoA pathway could not be de- tected, strongly indicating that in D. hydrogenophilus, the RTCA cycle is the major pathway for CO, fixation (54). In H. thermophilus, an aerobic, thermophilic, obligately chemo- lithoautotrophic, hydrogen-oxidizing bacterium, enzymatic characterization has been performed on ATP-citrate lyase, pyruvate synthase, and two structurally distinct 2-oxoglutar- ate synthases. The ATP-citrate lyase from H. thermophilus is an enzyme consisting of six identical 43 kDa subunits (55), much smaller in size compared to the enzymes from mammalian cells and Chlorobium (see below). Pyruvate synthase (56) and a two-subunit 2-oxoglutarate synthase (57, 58) exhibited activity with cofactors such as FAD, FMN, or ferredoxin from the native host cells. Interestingly, an additional five-subunit 2-oxoglutarate synthase has re- cently been identified from H. thermophilus (59). This en- zyme, along with the two-subunit one, displays strict sub- strate specificity towards 2-oxoglutarate. As both genes are expressed in the cells, it will be of interest to elucidate the different contributions, if any, of the two enzymes in the RTCA cycle.

Enzymes of the RTCA cycle from green sulfur bacteria, such as C. limicola and C. tepidum have also been ex- amined. In C. tepidum, one of the key enzymes of the cycle, ATP-citrate lyase, has been purified and characterized (60). The complete genome sequence of C. tepidum has recently been determined, and this will surely provide valuable in- formation in future studies on the RTCA cycle. In C. limi- cola, genes encoding ATP-citrate lyase and isocitrate dehy- drogenase have been isolated, and the recombinant proteins have been characterized. In contrast to previously reported enzymes, ATP-citrate lyase from C. limicola is composed of two polypeptides (a: 66 kDa; p: 44 kDa) encoded by two genes, aclA and aclB (61). The catalytic residue that is ini- tially phosphorylated by ATP is located on the CL subunit, but both subunits are essential for catalytic activity. The en- zyme catalyzed the cleavage of citrate in an ATP-, CoA-, and Mg2’-dependent manner, and did not exhibit citrate synthase activity. The enzyme displayed typical Michaelis- Menten kinetics toward ATP, with an apparent K,,, value of 0.21 mM. However, strong negative cooperativity was ob- served with respect to citrate binding. Although the dissoci- ation constant of the first citrate molecule was O.O57mM, binding of the first citrate molecule to the enzyme drasti- cally decreased the affinity of the enzyme for the second

molecule by a factor of 23. ADP was a competitive inhibitor of ATP, with a Ki value of 0.037 mM. These kinetic features indicate that ATP-citrate lyase can regulate both the direc- tion and flux of the RTCA cycle in C. limicola (62).

Isocitrate dehydrogenase of C. limicola (CLIDH) is an NADP-dependent, monomeric enzyme of 80 kDa, structur- ally distinct from the dimeric NADP-dependent enzymes predominantly found in the tricarboxylic acid cycle of bac- teria or eukaryotic mitochondria (63). The kinetic properties of Cl-IDH and the dimeric, NADP-dependent isocitrate de- hydrogenase from Saccharomyces cerevisiae (SC-IDH) were found to be similar at their respective optimum pH values. However, at neutral pH, in contrast to the 20% activity of SC-IDH toward carboxylation as compared with that toward decarboxylation, the activities of Cl-IDH for both directions were nearly equivalent, suggesting that Cl-IDH possesses more favorable properties as a CO,-fixation enzyme at the physiological pH. Among various intermediates, oxaloace- tate proved to be a competitive inhibitor of 2-oxoglutarate (Ki: 0.35 mM) in the carboxylation reaction by Cl-IDH, a feature not found in the enzyme from S. cerevisiae (62).

Examination of ATP-citrate lyase and isocitrate dehydro- genase from C. limicola has revealed that these enzymes possess interesting biochemical features not found in pre- viously characterized enzymes. The results also imply the presence of post-translational regulation of the RTCA cycle depending on energy supply and accumulation of metabolic intermediates (Fig. 6). Future biochemical examination of the remaining enzymes, along with in vivo studies, will be necessary to understand how the RTCA cycle is regulated in order to sustain efficient growth in autotrophic microorga- nisms.

FUTURE PERSPECTIVES

This review has mainly dealt with two carbon dioxide fixation pathways found in microbes, the Calvin-Benson- Bassham cycle and the reductive tricarboxylic acid cycle. The distribution of the four known carbon dioxide fixing pathways is summarized in Table 1. Intriguingly, although several active Rubiscos have been identified, there is still a lack of evidence that supports the presence of a functional Calvin cycle in Archaea. Furthermore, among Bacteria, it seems that the Calvin cycle is not present in strict an- aerobes. In contrast, the RTCA cycle has been identified in both Bacteria and Archaea, and in both the anaerobic Chlo- rob&m and aerobic Hydrogenobacter. While the Calvin cycle, the RTCA cycle, and the 3-hydroxypropionate cycle are present in both photo- and chemoautotrophic micro- organisms, the acetyl-CoA pathway is confined to chemo- autotrophs.

Is there a fifth, or even a sixth pathway that can support autotrophic growth? With the variety of known carbon di- oxide fixing enzymes, including 2-oxoglutarate synthase, isocitrate dehydrogenase, phosphoenolpyruvate carboxy- lase, pyruvate carboxylase, phosphoenolpyruvate carboxy- kinase, pyruvate synthase, formate dehydrogenase, carbon monoxide dehydrogenase, and others, one could easily de- sign a feasible pathway capable of supporting autotrophic growth. Taking into account the ever-increasing number of

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VOL. 94.2002

Amino acids *

MICROBIAL CO, FIXATION 503

Sugars

f f

COOH

PhosphoenoIpyruvate \ COOH

k-0

Succinyl-GoA

Amino acids

FIG. 6. Reductive tricarboxylic acid cycle of ChIorobium limicola. Regulatory (inhibition) mechanisms suggested by in vitro biochemical analyses of ATP-citrate lyase and isocitrate dehydrogenase are included. Enzymes are numbered as follows: 1, ATP-citrate lyase (ACL); 2, malate

dehydrogenase; 3, fumarase; 4, succinate dehydrogenase; 5, acetyl-CoA: succinate CoA transferase (estimated from enzymatic examination of D. hydrogenophilus); 6,2-oxoglutarate synthase; 7, isocitrate dehydrogenase (IDH); 8, aconitase; 9, pyruvate synthase; 10, phosphoenolpyruvate syn- thetase; 11, phosphoenolpyruvate carboxylase. Reactions catalyzed by IDH and ACL are indicated by thick arrows.

TABLE 1. Representative distribution of the four known carbon dioxide fixation pathways

Pathway Bacteria Archaea

Calvin-Benson-Bassham cycle Cyanobacteria Synechococcus, Anacystis, Anabaena

Purple nonsulfur bacteria Rhodobacter, Rhodospirillum

Purple sulfur bacteria Chromatium

Some hydrogen-oxidizers Ralstonia, Hydrogenovibrio

Some sulfur-oxidizers Thiobacillus

Nitrite-oxidizers Nitrobacter

Ammonia-oxidizers Nitrosomonas, Nitrosococcus, Nitrosospira

Acetyl-CoA pathway Acetogens Clostridium, Acetobacterium

Some sulfate-reducers Desulfobacterium, Desulfovibrio

Methanogens Methanobacterium, Methanosarcina, Methanococcus

Some hydrogen oxidizers Archaeoglobus, Ferroglobus

3-Hydroxypropionate cycle

Reductive tricarboxylic acid cycle

Green nonsulfur bacteria Chloroflexus

Green sulfur bacteria Chlorobium

Some hydrogen-oxidizers Hydrogenobacter, Aqutfex

Some sulfate-reducers Desulfobacter

Some sulfur-oxidizers Acidianus, Metallosphaera

Some sultirr-reducers Thermoproteus

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504 ATOM1 J. BIOSCL BIOENG.,

microorganisms, particularly the extremophiles, that are be- ing isolated each year, the chances of discovering a novel pathway, or at least a modified one, are not at all low.

ACKNOWLEDGMENTS 15.

The biochemical studies on archaeal Rubisco from Z kodakaraensis KODl and the RTCA cycle from C. limicola were carried out with Drs. Satoshi Ezaki, Toshiaki Fukui, Tamotsu Kanai, Norihiro Maeda, and Tadayoshi Kanao, Mr. Tsukuru Kishimoto and Ms. Mineko Kawamura in the laboratory of Prof. Tadayuki Imanaka, Graduate School of Engineering, Kyoto Uni- versity. Structural studies on the archaeal Rubisco were carried out in a fruitful collaboration with Dr. Ken Kitano and Prof. Kunio Miki, Graduate School of Science, Kyoto University.

16.

17.

18.

19.

J.M., and Tabita, F. R: Deduced amino acid sequence, functional expression, and unique enzymatic properties of the Form I and Form II ribulose bisphosphate carboxylase/oxy- genase from the chemoautotrophic bacterium Thiobacillus denitrzjicans. J. Bacterial., 178,347-356 (1996). Hayashi, N. R., Arai, H., Kodama, T., and Igarashi, Y.: The cbbQ genes, located downstream of the Form I and Form II RubisCO genes, affect the activity of both RubisCOs. Bio- them. Biophys. Res. Commun., 265,177-l 83 (1999). Morse, D., Salois, P., Markovic, P., and Hastings, J. W.: A nuclear-encoded Form II RuBisCO in dinoflagellates. Science, 268, 1622-1624 (1995). Schneider, G., Lindqvist, Y., and BrBndBn, C-I.: RUBISCO: structure and mechanism. Annu. Rev. Biophys. Biomol. Struct., 21, 119-143 (1992). Mann, C. C.: Genetic engineers aim to soup up crop photo- synthesis. Science, 283, 3 14-3 16 (1999). Whitney, S. M. and Andrews, T. J.: Plastome-encoded bacterial ribulose-1,5_bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in tobacco. Proc. Natl. Acad. Sci. USA, 98, 14738-14743 (2001). Read, B. A. and Tabita, F. R: High substrate specificity fac- tor ribulose bisphosphate carboxylase/oxygenase from eu- karyotic marine algae and properties of recombinant cyano- bacterial Rubisco containing “Algal” residue modifications. Arch. Biochem. Biophys., 312,210-218 (1994). Uemura, K., Anwaruzzaman, Miyachi, S., and Yokota, A.: Ribulose-1,5_bisphosphate carboxylase/oxygenase from ther- mophilic red algae with a strong specificity for CO, fixation. Biochem. Biophys. Res. Commun., 233,568-571 (1997). Harpel, M. R., Larimer, F. W., and Hartman, F. C.: Multi- faceted roles of Lysl66 of ribulose-bisphosphate carboxy- lase/oxygenase as discerned by product analysis and chemical rescue of site-directed mutants. Biochemistry, 41, 1390-I 397 (2002). Horken, K.M. and Tabita, F. R: The “green” Form I ribulose 1,5_bisphosphate carboxylase/oxygenase from the nonsulfur purple bacterium Rhodobacter capsulatus. J. Bac- teriol., 181,3935-3941 (1999). Wang, X., Falcone, D. L., and Tabita, F.R.: Reductive pentose phosphate-independent CO, fixation in Rhodobacter sphaeroides and evidence that ribulose bisphosphate carboxy- lase/oxygenase activity serves to maintain the redox balance ofthe cell. J. Bacterial., 175, 3372-3379 (1993). Hansen, S., Vollan, V. B., Hough, E., and Andersen, K.: The crystal structure of Rubisco from Alcaligenes eutrophus reveals a novel central eight-stranded P-barrel formed by p- strands from four subunits. J. Mol. Biol., 288, 609-621 (1999). Bainbridge, G., Anralojc, P. J., Madgwick, P. J., Pitts, J. E., and Parry, M. A.: Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxy- genase from Anacystis nidulans. Biochem. J., 336, 387-393 (1998). Newman, J. and Gutteridge, S.: Structure of an effector- induced inactivated state of ribulose 1,5_bisphosphate car- boxylase/oxygenase: the binary complex between enzyme and xylulose 1,5_bisphosphate. Structure, 2,495-502 (1994). Newman, J. and Gutteridge, S.: The X-ray structure of Synechococcus ribulose-bisphosphate carboxylaseioxygenase- activated quatemary complex at 2.2-A resolution. J. Biol. Chem., 268,25876-25886 (1993). Schneider, G., Lindqvist, Y., BriindBn, C-I., and Lorimer, G.: Three-dimensional structure of ribulose-1,5_bisphosphate carboxylaseloxygenase Tom Rhodospirillum rubrum at 2.9 8, resolution. EMBO .I., 5,3409-3415 (1986). Paoli, G. C., Vichivanives, P., and Tab&a, F. R.: Physiologi- cal control and regulation of the Rhodobacter capsulatus ebb operons. J. Bacterial., 180,4258%4269 (1998).

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

REFERENCES

Shively, J. M., van Keulen, G., and Meijer, W. G.: Some- thing from almost nothing: carbon dioxide fixation in chemo- autotrophs. Annu. Rev. Microbial., 52, 191-230 (1998). Wood, H. G., Ragsdale, S. W., and Pezacka, E.: The acetyl- CoA pathway: a newly discovered pathway of autotrophic growth. Trends Biol. Sci., 11, 14-18 (1986). Ragsdale, S. W.: Enzymology of the acetyl-CoA pathway of CO, fixation. Crit. Rev. Biochem. Mol. Biol., 26, 261-300 (1991). Herter, S., Fuchs, G., Bather, A., and Eisenreich, W.: A bicyclic autotrophic CO, fixation pathway in ChZoroJlexus aurantiacus. J. Biol. Chem., 277,20277-20283 (2002). Evans, M. C., Buchanan, B. B., and Arnon, D. I.: A new ferredoxin-dependent carbon reduction cycle in a photosyn- thetic bacterium. Proc. Natl. Acad. Sci. USA, 55, 928-934 (1966). Buchanan, B. B. and Arnon, D. I.: A reverse KREBS cycle in photosynthesis: consensus at last. Photosynth. Res., 24, 47-53 (1990). Hugler, M., Menendez, C., Schagger, H., and Fuchs, G.: Malonyl-coenzyme A reductase from Chlorojlexus aurantia- cus, a key enzyme of the 3-hydroxypropionate cycle for auto- trophic CO, fixation. J. Bacterial., 184,2404-2410 (2002). Alber, B. E. and Fuchs, G.: Propionyl-coenzyme A synthase from ChloroJlexus aurantiacus, a key enzyme of the 3-hy- droxypropionate cycle for autotrophic CO, fixation. J. Biol. Chem., 277, 12137-12143 (2002). Hartman, F. C. and Harpel, M. R.: Structure, function, reg- ulation, and assembly of D-ribulose-1 $bisphosphate carbox- ylase/oxygenase. Annu. Rev. Biochem., 63, 197-234 (1994). Watson, G. M. F. and Tabita, F. R.: Microbial ribulose 1,5- bisphosphate carboxylaseioxygenase: a molecule for phylo- genetic and enzymological investigation. FEMS Microbial. Lett., 146, 13-22 (1997). Tabita, F. R: The biochemistry and metabolic regulation of carbon metabolism and CO, fixation in purple bacteria, p. 885-914. In Blankenship, R. E., Madigan, M.T., and Bauer, C. E. (ed.), Anoxygenic photosynthetic bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands (1995). Hartman, F. C., Stringer, C. D., and Lee, E. H.: Complete primary structure of iibulose carboxylaseioxygenase from Rhodoswirillum rubrum. Arch. Biochem. Bioohvs.. 232. 280- 295 (19’84).

III ,

Paoli, G. C., Morgan, N. S., Tabita, F. R., and Shively, J.M.: Expression of the cbbLcbbS and cbbM genes and distinct organization of the ebb Calvin cycle structural genes of Rhodobacter capsulatus. Arch. Microbial., 164, 396405 (1995). Hernandez, J. M., Baker, S. H., Lorbach, S. C., Shively,

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

Page 9: Microbial Enzymes Involved in Carbon Dioxide Fixation

VOL. 94,2002 MICROBIAL CO, FIXATION 505

3 1. Tichi, M. A. and Tabita, F. R.: Maintenance and control of redox poise in Rhodobacter capsulatus strains deficient in the Calvin-Benson-Bassham pathway. Arch. Microbial., 174, 322-333 (2000).

32. Dubbs, J. M., Bird, T. H., Bauer, C. E., and Tabita, F. R.: Interaction of CbbR and RegA* transcription regulators with the Rhodobacter sphaeroides ebb, promoter-operator region. J. Biol. Chem., 275, 19224-19230 (2000).

33. Vichivanives, P., Bird, T. H., Bauer, C. E., and Tabita, F. FL: Multiple regulators and their interactions in viva and in vitro with the ebb regulons of Rhodobacter capsulatus. J. Mol. Biol., 300, 1079-1099 (2000).

34. Tichi, M. A. and Tabita, F. R.: Metabolic signals that lead to control of CBB gene expression in Rhodobacter capsulatus. J. Bacterial., 184, 1905-1915 (2002).

35. Ezaki, S., Maeda, N., Kishimoto, T., Atomi, H., and Imanaka, T.: Presence of a structurally novel type ribulose- bisphosphate carboxylase/oxygenase in the hyperthermo- philic archaeon, Pyrococcus kodakaraensis KODl. J. Biol. Chem., 274,5078-5082 (1999).

36. Maeda, N., Kitano, K., Fukui, T., Ezaki, S., Atomi, H., Miki, K., and Imanaka, T.: Ribulose bisphosphate carboxy- lase/oxygenase from the hyperthermophilic archaeon Pyro- coccus kodakaraensis KODl is composed solely of large sub- units and forms a pentagonal structure. J. Mol. Biol., 293, 57- 66 (1999).

37. Atomi, H., Ezaki, S., and Imanaka, T.: Ribulose-1,5- bisphosphate carboxylasefoxygenase from Thermococcus kodakaraensis KODl. Methods Enzymol., 331, 353-365 (2001).

38. Kitano, K., Maeda, N., Fukui, T., Atomi, H., Imanaka, T., and M&i, K.: Crystal structure of a novel-type archaeal rubisco with pentagonal symmetry. Structure, 9, 473481 (2001).

39. Maeda, N., Kanai, T., Atomi, H., and Imanaka, T.: The unique pentagonal structure of an archaeal Rubisco is essen- tial for its high thermostability. J. Biol. Chem., 277, 31656- 3 1662 (2002).

40. Watson, G. M. F., Yu, J-P., and Tabita, F. R.: Unusual ribulose 1,5-bisphosphate carboxylaseioxygenase of anoxic archaea. J. Bacterial., 181, 1569-1575 (1999).

41. Morikawa, M., Izawa, Y., Rashid, N., Hoaki, T., and Imanaka, T.: Purification and characterization of a thermo- stable thiol protease from a newly isolated hyperthermophiic Pyrococcus sp. Appl. Environ. Microbial., 60, 4559-4566 (1994).

42. Hashimoto, H., Nishioka, M., Fujiwara, S., Takagi, M., Imanaka, T., Inoue, T., and Kai, Y.: Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyro- coccus kodakaraensis KODl. J. Mol. Biol., 306, 469477 (2001).

43. Nakatani, M., Ezaki, S., Atomi, H., and Imanaka, T.: A DNA ligase from a hyperthermophilic archaeon with unique cofactor specificity. J. Bacterial., 182,6424-6433 (2000).

44. Nakatani, M., Ezaki, S., Atomi, H., and Imanaka, T.: Sub- strate recognition and fidelity of strand joining by an archaeal DNA ligase. Eur. J. Biochem., 269,650-656 (2002).

45. Izumi, M., Fujiwara, S., Takagi, M., Fukui, K., and Imanaka, T.: Utilization of immobilized archaeal chapero- nin for enzyme stabilization. J. Biosci. Bioeng., 91, 3 16-3 18 (2001).

46. Tachibana, Y., Takaha, T., Fujiwara, S., Takagi, M., and Imanaka, T.: Acceptor specificity of cL-glucanotransferase from Pyrococcus kodakaraensis KODl, and synthesis of cycloamylose. J. Biosci. Bioeng., 90,406409 (2000).

47. Kuriki, T. and Imanaka, T.: The concept of the alpha amy- lase family; structural similarity and common catalytic mech- anism. J. Biosci. Bioeng., 87, 557-565 (1999).

48. Ezaki, S., Miyaoku, K., Nishi, K., Tanaka, T., Fujiwara, S.,

Takagi, M., Atomi, H., and Imanaka, T.: Gene analysis and enzymatic properties of thermostable P-glycosidase from Qrococcus kodakaraensis KODl. J. Biosci. Bioeng., 88, 130-135 (1999).

49. Tanaka, T., Fukui, T., and Imanaka, T.: Different cleavage specificities of the dual catalytic domains in chitinase from the hyperthermophilic archaeon Thermococcus kodakaraen- sis KODl. J. Biol. Chem., 276, 35629-35635 (2001).

50. Rashid, N., Imanaka, H., Kanai, T., Fukui, T., Atomi, H., and Imanaka, T.: A novel candidate for the true fiuctose- 1,6-bisphosphatase in archaea. J. Biol. Chem., 277, 30649- 30655 (2002).

51. Imanaka, H., Fukui, T., Atomi, H., and Imanaka, T.: Gene cloning and characterization of fructose- 1,6-bisphosphate aldolase from the hyperthermophilic archaeon Thermococ- cus kodakaraensis KODl. J. Biosci. Bioeng., 94, 237-243 (2002).

52. Hanson, T. E. and Tabita, F. R.: A ribulose-1,5-bisphos- phate carboxylase/oxygenase (RubisCO)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress. Proc. Natl. Acad. Sci. USA, 98,43974402 (2001).

53. Sintsov, N.V., Ivanovskii, R.N., and Kondrat’eva, E. N.: ATP-dependent citrate lyase in the green phototrophic bac- terium, Chlorobium limicola. Mikrobiologiia, 49, 5 14-5 16 (1980).

54. Schauder, R, Widdel, F., and Fuchs, G.: Carbon assimila- tion pathways in sulfate-reducing bacteria II. Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus. Arch. Microbial., 148,218-225 (1987).

55. Ishii, M., Igarashi, Y., and Kodama, T.: Purification and characterization of ATP : citrate lyase from Hydrogenobacter thermophilus TK-6. J. Bacterial., 171, 1788-1792 (1989).

56. Yoon, K. S., Ishii, M., Kodama, T., and Igarashi, Y.: Purifi- cation and characterization of pyruvate : ferredoxin oxido- reductase from Hydrogenobacter thermophilus TK-6. Arch. Microbial., 167,275-279 (1997).

57. Yoon, K. S., Ishii, M., Igarashi, Y., and Kodama, T.: Purifi- cation and characterization of 2-oxoglutarate : ferredoxin oxi- doreductase from a thermophilic, obligately chemolithoau- totrophic bacterium, Hydrogenobacter thermophilus TK-6. J. Bacterial., 178, 3365-3368 (1996).

58. Yun, N. R., Arai, H., Ishii, M., and Igarashi, Y.: The genes for anabolic 2-oxoglutarate: ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6. Biochem. Biophys. Res. Commun., 282,589-594 (2001).

59. Yun, N. R., Yamamoto, M., Arai, H., Ishii, M., and Igarashi, Y.: A novel five-subunit-type 2-oxoglutalate : ferre- doxin oxidoreductases from Hydrogenobacter thermophilus TK-6. Biochem. Biophys. Res. Commun., 292, 280-286 (2002).

60. Wahlund, T. M. and Tabita, F. R.: The reductive tricarbox- ylic acid cycle of carbon dioxide assimilation: initial studies and purification of ATP-citrate lyase from the green sulfur bacterium Chlorobium tepidum. J. Bacterial., 179,48594867 (1997).

61. Kanao, T., Fukui, T., Atomi, H., and Imanaka, T.: ATP- citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products. Eur. J. Biochem., 268, 1670-1678 (2001).

62. Kanao, T., Fukui, T., Atomi, H., and Imanaka, T.: Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyase. Eur. J. Biochem., 269,3409-3416 (2002).

63. Kanao, T., Kawamura, M., Fukui, T., Atomi, H., and Imanaka, T.: Characterization of isocitrate dehydrogenase from the green sulfur bacterium Chlorobium limicola. A carbon dioxide-fixing enzyme in the reductive tricarboxylic acid cycle. Eur. J. Biochem., 269, 1926-1931 (2002).