Properties of R-citramalyl-coenzyme A lyase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The autotrophic CO(2) fixation pathway (3-hydroxypropionate cycle) in Chloroflexus aurantiacus results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO(2) acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO(2)) to beta-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both S- and R-citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the S- and R-citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth. S-Citramalyl-CoA lyase activity was found to be due to l-malyl-CoA lyase/beta-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO(2) fixation pathway, forms acetyl-CoA and glyoxylate from l-malyl-CoA, and condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA. R-Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg(2) or Mn(2+) ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in C. aurantiacus. Genes similar to those involved in CO(2) fixation in C. aurantiacus, including an R-citramalyl-CoA lyase gene, were found in Roseiflexus sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the gamma-proteobacterium Congregibacter litoralis. This may indicate that part of the reactions may be involved in a different metabolic process.     

Properties of succinyl-coenzyme A:L-malate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic CO2 fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in CO2 fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large (alphabeta)n complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate --> succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor; the corresponding d-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.     

Properties of succinyl-coenzyme A:D-citramalate coenzyme A transferase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The phototrophic bacterium Chloroflexus aurantiacus uses the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. This cycle starts with acetyl-coenzyme A (CoA) and produces glyoxylate. Glyoxylate is an unconventional cell carbon precursor that needs special enzymes for assimilation. Glyoxylate is combined with propionyl-CoA to beta-methylmalyl-CoA, which is converted to citramalate. Cell extracts catalyzed the succinyl-CoA-dependent conversion of citramalate to acetyl-CoA and pyruvate, the central cell carbon precursor. This reaction is due to the combined action of enzymes that were upregulated during autotrophic growth, a coenzyme A transferase with the use of succinyl-CoA as the CoA donor and a lyase cleaving citramalyl-CoA to acetyl-CoA and pyruvate. Genomic analysis identified a gene coding for a putative coenzyme A transferase. The gene was heterologously expressed in Escherichia coli and shown to code for succinyl-CoA:d-citramalate coenzyme A transferase. This enzyme, which catalyzes the reaction d-citramalate + succinyl-CoA --> d-citramalyl-CoA + succinate, was purified and studied. It belongs to class III of the coenzyme A transferase enzyme family, with an aspartate residue in the active site. The homodimeric enzyme composed of 44-kDa subunits was specific for succinyl-CoA as a CoA donor but also accepted d-malate and itaconate instead of d-citramalate. The CoA transferase gene is part of a cluster of genes which are cotranscribed, including the gene for d-citramalyl-CoA lyase. It is proposed that the CoA transferase and the lyase catalyze the last two steps in the glyoxylate assimilation route.     

Autotrophic CO2 Fixation by Chloroflexus aurantiacus: Study of Glyoxylate Formation and Assimilation via the 3-Hydroxypropionate Cycle

In the facultative autotrophic organism Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium, the Calvin cycle does not appear to be operative in autotrophic carbon assimilation. An alternative cyclic pathway, the 3-hydroxypropionate cycle, has been proposed. In this pathway, acetyl coenzyme A (acetyl-CoA) is assumed to be converted to malate, and two CO(2) molecules are thereby fixed. Malyl-CoA is supposed to be cleaved to acetyl-CoA, the starting molecule, and glyoxylate, the carbon fixation product. Malyl-CoA cleavage is shown here to be catalyzed by malyl-CoA lyase; this enzyme activity is induced severalfold in autotrophically grown cells. Malate is converted to malyl-CoA via an inducible CoA transferase with succinyl-CoA as a CoA donor. Some enzyme activities involved in the conversion of malonyl-CoA via 3-hydroxypropionate to propionyl-CoA are also induced under autotrophic growth conditions. So far, no clue as to the first step in glyoxylate assimilation has been obtained. One possibility for the assimilation of glyoxylate involves the conversion of glyoxylate to glycine and the subsequent assimilation of glycine. However, such a pathway does not occur, as shown by labeling of whole cells with [1,2-(13)C(2)]glycine. Glycine carbon was incorporated only into glycine, serine, and compounds that contained C(1) units derived therefrom and not into other cell compounds.     

A bicyclic autotrophic CO2 fixation pathway in Chloroflexus aurantiacus

Phototrophic CO(2) assimilation by the primitive, green eubacterium Chloroflexus aurantiacus has been shown earlier to proceed in a cyclic mode via 3-hydroxypropionate, propionyl-CoA, succinyl-CoA, and malyl-CoA. The metabolic cycle could be closed by cleavage of malyl-CoA affording glyoxylate (the primary CO(2) fixation product) with regeneration of acetyl-CoA serving as the starter unit of the cycle. The pathway of glyoxylate assimilation to form gluconeogenic precursors has not been elucidated to date. We could now show that the incubation of cell extract with a mixture of glyoxylate and [1,2,3-(13)C(3)]propionyl-CoA afforded erythro-beta-[1,2,2'-(13)C(3)]methylmalate and [1,2,2'-(13)C(3)]citramalate. Similar experiments using a partially purified protein fraction afforded erythro-beta-[1,2,2'-(13)C(3)]methylmalyl-CoA and [1,2,2'-(13)C(3)]mesaconyl-CoA. Cell extracts of C. aurantiacus were also shown to catalyze the conversion of citramalate into pyruvate and acetyl-CoA in a succinyl-CoA-dependent reaction. The data suggest that glyoxylate obtained by the cleavage of malyl-CoA can be utilized by condensation with propionyl-CoA affording erythro-beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. This reaction sequence regenerates acetyl-CoA, which serves as the precursor of propionyl-CoA in the 3-hydroxypropionate cycle. Autotrophic CO(2) fixation proceeds by combination of the 3-hydroxypropionate cycle with the methylmalyl-CoA cycle. The net product of that bicyclic autotrophic CO(2) fixation pathway is pyruvate serving as an universal building block for anabolic reactions.     

L-malyl-coenzyme A/beta-methylmalyl-coenzyme A lyase is involved in acetate assimilation of the isocitrate lyase-negative bacterium Rhodobacter capsulatus

Cell extracts of Rhodobacter capsulatus grown on acetate contained an apparent malate synthase activity but lacked isocitrate lyase activity. Therefore, R. capsulatus cannot use the glyoxylate cycle for acetate assimilation, and a different pathway must exist. It is shown that the apparent malate synthase activity is due to the combination of a malyl-coenzyme A (CoA) lyase and a malyl-CoA-hydrolyzing enzyme. Malyl-CoA lyase activity was 20-fold up-regulated in acetate-grown cells versus glucose-grown cells. Malyl-CoA lyase was purified 250-fold with a recovery of 6%. The enzyme catalyzed not only the reversible condensation of glyoxylate and acetyl-CoA to L-malyl-CoA but also the reversible condensation of glyoxylate and propionyl-CoA to beta-methylmalyl-CoA. Enzyme activity was stimulated by divalent ions with preference for Mn(2+) and was inhibited by EDTA. The N-terminal amino acid sequence was determined, and a corresponding gene coding for a 34.2-kDa protein was identified and designated mcl1. The native molecular mass of the purified protein was 195 +/- 20 kDa, indicating a homohexameric composition. A homologous mcl1 gene was found in the genomes of the isocitrate lyase-negative bacteria Rhodobacter sphaeroides and Rhodospirillum rubrum in similar genomic environments. For Streptomyces coelicolor and Methylobacterium extorquens, mcl1 homologs are located within gene clusters implicated in acetate metabolism. We therefore propose that L-malyl-CoA/beta-methylmalyl-CoA lyase encoded by mcl1 is involved in acetate assimilation by R. capsulatus and possibly other glyoxylate cycle-negative bacteria.     

Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation

The 3-hydroxypropionate cycle has been proposed as a new autotrophic CO(2) fixation pathway for the phototrophic green non-sulfur eubacterium Chloroflexus aurantiacus and for some chemotrophic archaebacteria. The cycle requires the reductive conversion of the characteristic intermediate 3-hydroxypropionate to propionyl-CoA. The specific activity of the 3-hydroxypropionate-, CoA-, K(+)-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.09 micromol min(-1) mg(-1) protein, which was 2-fold down-regulated in heterotrophically grown cells. Unexpectedly, a single enzyme catalyzes the entire reaction sequence: 3-hydroxypropionate + MgATP + CoA + NADPH + H(+) --> propionyl-CoA + MgAMP + PP(i) + NADP(+) + H(2)O. The enzyme was purified 30-fold to near homogeneity and has a very large native molecular mass between 500 and 800 kDa, with subunits of about 185 kDa as judged by SDS-PAGE, suggesting a homotrimeric or homotetrameric structure. Upon incubation of this new enzyme, termed propionyl-CoA synthase, with the proteinase trypsin, the NADPH oxidation function of the enzyme was lost, whereas the enzyme still activated 3-hydroxypropionate to its CoA-thioester and dehydrated it to acrylyl-CoA. SDS-PAGE revealed that the subunits of propionyl-CoA synthase had been cleaved once and the N-terminal amino acid sequences of the two trypsin digestion products were determined. Two parts of the gene encoding propionyl-CoA synthase (pcs) were identified on two contigs of an incomplete genome data base of C. aurantiacus, and the sequence of the pcs gene was completed. Propionyl-CoA synthase is a natural fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA hydratase, and an enoyl-CoA reductase, the reductase domain containing the trypsin cleavage site. Similar polyfunctional large enzymes are common in secondary metabolism (e.g. polyketide synthases) but rare in primary metabolism (e.g. eukaryotic type I fatty acid synthase). These results lend strong support to the operation of the proposed pathway in autotrophic CO(2) fixation.     

13C-NMR study of autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus.

The unresolved autotrophic CO2 fixation pathways in the sulfur-reducing Archaebacterium Thermoproteus neutrophilus and in the phototrophic Eubacterium Chloroflexus aurantiacus have been investigated. Autotrophically growing cultures were labelled with [1,4-13C1]succinate, and the 13C pattern in cell constituents was determined by 1H- and 13C-NMR spectroscopy of purified amino acids and other cell constituents. In both organisms succinate contributed to less than 10% of cell carbon, the major part of carbon originated from CO2. All cell constituents became 13C-labelled, but different patterns were observed in the two organisms. This proves that two different cyclic CO2 fixation pathways are operating in autotrophic carbon assimilation in both of which succinate is an intermediate. The 13C-labelling pattern in T. neutrophilus is consistent with the operation of a reductive citric acid cycle and rules out any other known autotrophic CO2 fixation pathway. Surprisingly, the proffered [1,4-13C1]succinate was partially converted to double-labelled [3,4-13C2]glutamate, but not to double-labelled aspartate. These findings suggest that the conversion of citrate to 2-oxoglutarate is readily reversible under the growth conditions used, and a reversible citrate cleavage reaction is proposed. The 13C-labelling pattern in C. aurantiacus disagrees with any of the established CO2 fixation pathways; it therefore demands a novel autotrophic CO2 fixation cycle in which 3-hydroxypropionate and succinate are likely intermediates. The bacterium excreted substantial amounts of 3-hydroxypropionate (5 mM) and succinate (0.5 mM) at the end of autotrophic growth. Autotrophically grown Chloroflexus cells contained acetyl-CoA carboxylase and propionyl-CoA carboxylase activity. These enzymes are proposed to be the main CO2-fixing enzymes resulting in malonyl-CoA and methylmalonyl-CoA formation; from these carboxylation products 3-hydroxypropionate and succinate, respectively, can be formed.     

Autotrophic carbon dioxide fixation inAcidianus brierleyi

The autotrophic CO₂ fixation pathway inAcidianus brierleyi, a facultatively anaerobic thermoacidophilic archaebacterium, was investigated by measuring enzymatic activities from autotrophic, mixotrophic, and heterotrophic cultures. Contrary to the published report that the reductive tricarboxylic acid cycle operates inA. brierleyi, the enzymatic activity of ATP:citrate lyase, the key enzyme of the cycle, was not detected. Instead, activities of acetyl-CoA carboxylase and propionyl-CoA carboxylase, key enzymes of the 3-hydroxypropionate cycle, were detected only whenA. brierleyi was growing autotrophically. We conclude that a modified 3-hydroxypropionate pathway operates inA. brierleyi.Abbreviations TCA tricarboxylic acid - BV Benzyl viologen     

Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides

Organisms, which grow on organic substrates that are metabolized via acetyl-CoA, are faced with the problem to form all cell constituents from this C(2)-unit. The problem was solved by the seminal work of Kornberg and is known as the glyoxylate cycle. However, many bacteria are known to not contain isocitrate lyase, the key enzyme of this pathway. This problem was addressed in acetate-grown Rhodobacter sphaeroides. An acetate-minus mutant identified by transposon mutagenesis was affected in the gene for beta-ketothiolase forming acetoacetyl-CoA from two molecules of acetyl-CoA. This enzyme activity was missing in this mutant, which grew on acetoacetate and on acetate plus glyoxylate. A second acetate/acetoacetate-minus mutant was affected in the gene for a putative mesaconyl-CoA hydratase, an enzyme which catalyses the hydration of mesaconyl-CoA to beta-methylmalyl-CoA. Beta-methylmalyl-CoA is further cleaved into glyoxylate and propionyl-CoA. These results as well as identification of acetate-upregulated proteins by two-dimensional gel electrophoresis lead to the proposal of a new pathway for acetate assimilation. In a first part, affected by the mutations, two molecules of acetyl-CoA and one molecule CO(2) are converted via acetoacetyl-CoA and mesaconyl-CoA to glyoxylate and propionyl-CoA. In a second part glyoxylate and propionyl-CoA are converted with another molecule of acetyl-CoA and CO(2) to l-malyl-CoA and succinyl-CoA.     

Mesaconyl-coenzyme A hydratase, a new enzyme of two central carbon metabolic pathways in bacteria

The coenzyme A (CoA)-activated C5-dicarboxylic acids mesaconyl-CoA and beta-methylmalyl-CoA play roles in two as yet not completely resolved central carbon metabolic pathways in bacteria. First, these compounds are intermediates in the 3-hydroxypropionate cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus, a phototrophic green nonsulfur bacterium. Second, mesaconyl-CoA and beta-methylmalyl-CoA are intermediates in the ethylmalonyl-CoA pathway for acetate assimilation in various bacteria, e.g., in Rhodobacter sphaeroides, Methylobacterium extorquens, and Streptomyces species. In both cases, mesaconyl-CoA hydratase was postulated to catalyze the interconversion of mesaconyl-CoA and beta-methylmalyl-CoA. The putative genes coding for this enzyme in C. aurantiacus and R. sphaeroides were cloned and heterologously expressed in Escherichia coli, and the proteins were purified and studied. The recombinant homodimeric 80-kDa proteins catalyzed the reversible dehydration of erythro-beta-methylmalyl-CoA to mesaconyl-CoA with rates of 1,300 micromol min(-1) mg protein(-1). Genes coding for similar enzymes with two (R)-enoyl-CoA hydratase domains are present in the genomes of Roseiflexus, Methylobacterium, Hyphomonas, Rhodospirillum, Xanthobacter, Caulobacter, Magnetospirillum, Jannaschia, Sagittula, Parvibaculum, Stappia, Oceanicola, Loktanella, Silicibacter, Roseobacter, Roseovarius, Dinoroseobacter, Sulfitobacter, Paracoccus, and Ralstonia species. A similar yet distinct class of enzymes containing only one hydratase domain was found in various other bacteria, such as Streptomyces species. The role of this widely distributed new enzyme is discussed.     

Presence of Acetyl Coenzyme A (CoA) Carboxylase and Propionyl-CoA Carboxylase in Autotrophic Crenarchaeota and Indication for Operation of a 3-Hydroxypropionate Cycle in Autotrophic Carbon Fixation

The pathway of autotrophic CO₂ fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO₂-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either ¹⁴CO₂ or [¹⁴C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO₂ fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.     

Autotrophic CO<Subscript>2</Subscript> fixation pathways in archaea (Crenarchaeota)

Representative autotrophic and thermophilic archaeal species of different families of Crenarchaeota were examined for key enzymes of the known autotrophic CO(2) fixation pathways. Pyrobaculum islandicum ( Thermoproteaceae) contained key enzymes of the reductive citric acid cycle. This finding is consistent with the operation of this pathway in the related Thermoproteus neutrophilus. Pyrodictium abyssi and Pyrodictium occultum ( Pyrodictiaceae) contained ribulose 1,5-bisphosphate carboxylase, which was active in boiling water. Yet, phosphoribulokinase activity was not detectable. Operation of the Calvin cycle remains to be demonstrated. Ignicoccus islandicus and Ignicoccus pacificus ( Desulfurococcaceae) contained pyruvate oxidoreductase as potential carboxylating enzyme, but apparently lacked key enzymes of known pathways; their mode of autotrophic CO(2) fixation is at issue. Metallosphaera sedula, Acidianus ambivalens and Sulfolobus sp. strain VE6 ( Sulfolobaceae) contained key enzymes of a 3-hydroxypropionate cycle. This finding is in line with the demonstration of acetyl-coenzyme A (CoA) and propionyl-CoA carboxylase activities in the related Acidianus brierleyi and Sulfolobus metallicus. Enzymes of central carbon metabolism in Metallosphaera sedula were studied in more detail. Enzyme activities of the 3-hydroxypropionate cycle were strongly up-regulated during autotrophic growth, supporting their role in CO(2) fixation. However, formation of acetyl-CoA from succinyl-CoA could not be demonstrated, suggesting a modified pathway of acetyl-CoA regeneration. We conclude that Crenarchaeota exhibit a mosaic of three or possibly four autotrophic pathways. The distribution of the pathways so far correlates with the 16S-rRNA-based taxa of the Crenarchaeota.     

Malonyl-coenzyme A reductase in the modified 3-hydroxypropionate cycle for autotrophic carbon fixation in archaeal Metallosphaera and Sulfolobus spp

Autotrophic members of the Sulfolobales (Crenarchaeota) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the CO2 fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate CO2 into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum Sulfolobus tokodaii. The Sulfolobus gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + H+ --> malonate-semialdehyde + CoA + NADP+. In its native state it is associated with small RNA. Its activity was stimulated by Mg2+ and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH (Km = 25 microM) and malonyl-CoA (Km = 40 microM). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonyl-CoA reductase from Chloroflexus aurantiacus. This shows that the autotrophic pathway in Chloroflexus and Sulfolobaceae has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.     

Malonyl-coenzyme A reductase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO(2) fixation

The 3-hydroxypropionate cycle is a new autotrophic CO(2) fixation pathway in Chloroflexus aurantiacus and some archaebacteria. The initial step is acetyl-coenzyme A (CoA) carboxylation to malonyl-CoA by acetyl-CoA carboxylase, followed by NADPH-dependent reduction of malonyl-CoA to 3-hydroxypropionate. This reduction step was studied in Chloroflexus aurantiacus. A new enzyme was purified, malonyl-CoA reductase, which catalyzed the two-step reduction malonyl-CoA + NADPH + H(+) --> malonate semialdehyde + NADP(+) + CoA and malonate semialdehyde + NADPH + H(+) --> 3-hydroxypropionate + NADP(+). The bifunctional enzyme (aldehyde dehydrogenase and alcohol dehydrogenase) had a native molecular mass of 300 kDa and consisted of a single large subunit of 145 kDa, suggesting an alpha(2) composition. The N-terminal amino acid sequence was determined, and the incomplete gene was identified in the genome database. Obviously, the enzyme consists of an N-terminal short-chain alcohol dehydrogenase domain and a C-terminal aldehyde dehydrogenase domain. No indication of the presence of a prosthetic group was obtained; Mg(2+) and Fe(2+) stimulated and EDTA inhibited activity. The enzyme was highly specific for its substrates, with apparent K(m) values of 30 microM malonyl-CoA and 25 microM NADPH and a turnover number of 25 s(-1) subunit(-1). The specific activity in autotrophically grown cells was 0.08 micromol of malonyl-CoA reduced min(-1) (mg of protein)(-1), compared to 0.03 micromol min(-1) (mg of protein)(-1) in heterotrophically grown cells, indicating downregulation under heterotrophic conditions. Malonyl-CoA reductase is not required in any other known pathway and therefore can be taken as a characteristic enzyme of the 3-hydroxypropionate cycle. Furthermore, the enzyme may be useful for production of 3-hydroxypropionate and for a coupled spectrophotometric assay for activity screening of acetyl-CoA carboxylase, a target enzyme of potent herbicides.     

Purification and properties of malyl-coenzyme A lyase from Pseudomonas AM1

1. Malyl-CoA lyase was purified 20-fold from extracts of methanol-grown Pseudomonas AM1. 2. Preparations of the enzyme were essentially homogeneous by electrophoretic and ultracentrifugal criteria. 3. Malyl-CoA lyase has a molecular weight of 190000 determined from sedimentation-equilibrium data. 4. Within the range of compounds tested, malyl-CoA lyase is specific for (2S)-4-malyl-CoA or glyoxylate and acetyl-CoA or propionyl-CoA. 5. A bivalent cation is essential for activity, Mg(2+) or Co(2+) being most effective. 6. Malyl-CoA lyase is inhibited by (2R)-4-malyl-CoA and by some buffers, but thiol-group inhibitors are without effect. 7. Optimal activity was recorded at pH7.8. 8. An equilibrium constant of 4.7x10(-4)m was determined for the malyl-CoA cleavage reaction. 9. The Michaelis constants for the enzyme are: 4-malyl-CoA, 6.6x10(-5)m; acetyl-CoA, 1.5x10(-5)m; glyoxylate, 1.7x10(-3)m; Mg(2+), 1.2x10(-3)m.     

Rhodobacter sphaeroides uses a reductive route via propionyl coenzyme A to assimilate 3-hydroxypropionate

3-Hydroxypropionate is a product or intermediate of the carbon metabolism of organisms from all three domains of life. However, little is known about how carbon derived from 3-hydroxypropionate is assimilated by organisms that can utilize this C(3) compound as a carbon source. This work uses the model bacterium Rhodobacter sphaeroides to begin to elucidate how 3-hydroxypropionate can be incorporated into cell constituents. To this end, a quantitative assay for 3-hydroxypropionate was developed by using recombinant propionyl coenzyme A (propionyl-CoA) synthase from Chloroflexus aurantiacus. Using this assay, we demonstrate that R. sphaeroides can utilize 3-hydroxypropionate as the sole carbon source and energy source. We establish that acetyl-CoA is not the exclusive entry point for 3-hydroxypropionate into the central carbon metabolism and that the reductive conversion of 3-hydroxypropionate to propionyl-CoA is a necessary route for the assimilation of this molecule by R. sphaeroides. Our conclusion is based on the following findings: (i) crotonyl-CoA carboxylase/reductase, a key enzyme of the ethylmalonyl-CoA pathway for acetyl-CoA assimilation, was not essential for growth with 3-hydroxypropionate, as demonstrated by mutant analyses and enzyme activity measurements; (ii) the reductive conversion of 3-hydroxypropionate or acrylate to propionyl-CoA was detected in cell extracts of R. sphaeroides grown with 3-hydroxypropionate, and both activities were upregulated compared to the activities of succinate-grown cells; and (iii) the inactivation of acuI, encoding a candidate acrylyl-CoA reductase, resulted in a 3-hydroxypropionate-negative growth phenotype.     

Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation.

Autotrophic Archaea of the family Sulfolobaceae (Crenarchaeota) use a modified 3-hydroxypropionate cycle for carbon dioxide assimilation. In this cycle the ATP-dependent carboxylations of acetyl-CoA and propionyl-CoA to malonyl-CoA and methylmalonyl-CoA, respectively, represent the key CO2 fixation reactions. These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl-CoA and propionyl-CoA. The carboxylase was purified and characterized and the genes were cloned and sequenced. In contrast to the carboxylase of most other organisms, acetyl-CoA/propionyl-CoA carboxylase from M. sedula is active at 75 degrees C and is isolated as a stabile functional protein complex of 560 +/- 50 kDa. The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (alpha) and carboxytransferase (gamma), respectively, and a small 18.6 kDa biotin carrier protein (beta). These subunits probably form an (alpha beta gamma)4 holoenzyme. It has a catalytic number of 28 s-1 at 65 degrees C and at the optimal pH of 7.5. The apparent Km values were 0.06 mm for acetyl-CoA, 0.07 mm for propionyl-CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate. Acetyl-CoA/propionyl-CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes. Due to its stability the archaeal carboxylase may prove an ideal subject for further structural studies.     

3-Hydroxypropionyl-Coenzyme A Dehydratase and Acryloyl-Coenzyme A Reductase,Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in the Sulfolobales

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates in autotrophic CO₂ fixation in various Crenarchaea, as studied in some detail in Metallosphaera sedula. This cycle and the autotrophic 3-hydroxypropionate cycle in Chloroflexus aurantiacus have in common the conversion of acetyl-coenzyme A (CoA) and two bicarbonates via 3-hydroxypropionate to succinyl-CoA. Both cycles require the reductive conversion of 3-hydroxypropionate to propionyl-CoA. In M. sedula the reaction sequence is catalyzed by three enzymes. The first enzyme, 3-hydroxypropionyl-CoA synthetase, catalyzes the CoA- and MgATP-dependent formation of 3-hydroxypropionyl-CoA. The next two enzymes were purified from M. sedula or Sulfolobus tokodaii and studied. 3-Hydroxypropionyl-CoA dehydratase, a member of the enoyl-CoA hydratase family, eliminates water from 3-hydroxypropionyl-CoA to form acryloyl-CoA. Acryloyl-CoA reductase, a member of the zinc-containing alcohol dehydrogenase family, reduces acryloyl-CoA with NADPH to propionyl-CoA. Genes highly similar to the Metallosphaera CoA synthetase, dehydratase, and reductase genes were found in autotrophic members of the Sulfolobales. The encoded enzymes are only distantly related to the respective three enzyme domains of propionyl-CoA synthase from C. aurantiacus, where this trifunctional enzyme catalyzes all three reactions. This indicates that the autotrophic carbon fixation cycles in Chloroflexus and in the Sulfolobales evolved independently and that different genes/enzymes have been recruited in the two lineages that catalyze the same kinds of reactions.In the thermoacidophilic autotrophic crenarchaeum Metallosphaera sedula, CO₂ fixation proceeds via a 3-hydroxypropionate/4-hydroxybutyrate cycle (8, 23, 24, 28) (Fig. ​(Fig.1).1). A similar cycle may operate in other autotrophic members of the Sulfolobales and in mesophilic Crenarchaea (Cenarchaeum sp. and Nitrosopumilus sp.) of marine group I. The cycle uses elements of the 3-hydroxypropionate cycle that was originally discovered in the phototrophic bacterium Chloroflexus aurantiacus (11, 16, 17, 19, 20, 32, 33). It involves the carboxylation of acetyl-coenzyme A (CoA) to malonyl-CoA by the biotin-dependent acetyl-CoA carboxylase. Malonyl-CoA is reduced via malonate semialdehyde to 3-hydroxypropionate (1), which is further reductively converted to propionyl-CoA (3). Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by a propionyl-CoA carboxylase that is similar or identical to acetyl-CoA carboxylase. In fact, only one copy of the genes for the acetyl-CoA/propionyl-CoA carboxylase subunits is present in most Archaea, suggesting that this is a promiscuous enzyme that acts on both acetyl-CoA and propionyl-CoA (24). (S)-Methylmalonyl-CoA is epimerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other members of the Sulfolobales. Enzymes are the following: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionyl-CoA synthetase (3-hydroxypropionate-CoA ligase, AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinate semialdehyde reductase (NADPH); 12, 4-hydroxybutyryl-CoA synthetase (4-hydroxybutyrate-CoA ligase, AMP-forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14, crotonyl-CoA hydratase; 15, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase. The two steps of interest are highlighted.In Chloroflexus succinyl-CoA is converted to (S)-malyl-CoA, which is cleaved by (S)-malyl-CoA lyase to acetyl-CoA (thus regenerating the CO₂ acceptor molecule) and glyoxylate (16). Glyoxylate is assimilated into cell material by a yet not completely resolved pathway (37). In Metallosphaera succinyl-CoA is converted via 4-hydroxybutyrate to two molecules of acetyl-CoA (8), thus regenerating the starting CO₂ acceptor molecule and releasing another acetyl-CoA for biosynthesis. Hence, the 3-hydroxypropionate/4-hydroxybutyrate cycle (Fig. ​(Fig.1)1) can be divided into two parts. The first part transforms one acetyl-CoA and two bicarbonates into succinyl-CoA, and the second part converts succinyl-CoA to two acetyl-CoA molecules.The reductive conversion of 3-hydroxypropionate to propionyl-CoA requires three enzymatic steps: activation of 3-hydroxypropionate to its CoA ester, dehydration of 3-hydroxypropionyl-CoA to acryloyl-CoA, and reduction of acryloyl-CoA to propionyl-CoA. In C. aurantiacus these three steps are catalyzed by a single large trifunctional enzyme, propionyl-CoA synthase (2). This 200-kDa fusion protein consists of a CoA ligase, a dehydratase, and a reductase domain. Attempts to isolate a similar enzyme from M. sedula failed. Rather, a 3-hydroxypropionyl-CoA synthetase was found (3), suggesting that the other two reactions may also be catalyzed by individual enzymes.Here, we purified the missing enzymes 3-hydroxypropionyl-CoA dehydratase and acryloyl-CoA reductase from M. sedula, identified the coding genes in the genome of M. sedula and other members of the Sulfolobales, produced recombinant enzymes as proof of function, and studied the enzymes in some detail. A comparison with the respective domains of propionyl-CoA synthase from C. aurantiacus indicates that the conversion of 3-hydroxypropionate to propionyl-CoA via the 3-hydroxypropionate route has evolved independently in these two phyla.     

Characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, of Euglena gracilis

The glyoxylate cycle is a modified form of the tricarboxylic acid cycle, which enables organisms to synthesize carbohydrates from C2 compounds. In the protozoan Euglena gracilis, the key enzyme activities of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), are conferred by a single bifunctional protein named glyoxylate cycle enzyme (Euglena gracilis glyoxylate cycle enzyme [EgGCE]). We analyzed the enzymatic properties of recombinant EgGCE to determine the functions of its different domains. The 62-kDa N-terminal domain of EgGCE was sufficient to provide the MS activity as expected from an analysis of the deduced amino acid sequence. In contrast, expression of the 67-kDa C-terminal domain of EgGCE failed to yield ICL activity even though this domain was structurally similar to ICL family enzymes. Analyses of truncation mutants suggested that the N-terminal residues of EgGCE are critical for both the ICL and MS activities. The ICL activity of EgGCE increased in the presence of micro-molar concentrations of acetyl-coenzyme A (CoA). Acetyl-CoA also increased the activity in a mutant type EgGCE with a mutation at the acetyl-CoA binding site in the MS domain of EgGCE. This suggests that acetyl-CoA regulates the ICL reaction by binding to a site other than the catalytic center of the MS reaction.