L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus,a bifunctional enzyme involved in autotrophic CO(2) fixation

The 3-hydroxypropionate cycle is a bicyclic autotrophic CO(2) fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae (Archaea). The proposed pathway involves in a first cycle the conversion of acetyl-coenzyme A (acetyl-CoA) and two bicarbonates to L-malyl-CoA via 3-hydroxypropionate and propionyl-CoA; L-malyl-CoA is cleaved by L-malyl-CoA lyase into acetyl-CoA and glyoxylate. In a second cycle, glyoxylate and another molecule of propionyl-CoA (derived from acetyl-CoA and bicarbonate) are condensed by a putative beta-methylmalyl-CoA lyase to beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. The putative L-malyl-CoA lyase gene of C. aurantiacus was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Beta-methylmalyl-CoA lyase was purified from cell extracts of C. aurantiacus and characterized. We show that these two enzymes are identical and that both enzymatic reactions are catalyzed by one single bifunctional enzyme, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase. Interestingly, this enzyme works with two different substrates in two different directions: in the first cycle of CO(2) fixation, it cleaves L-malyl-CoA into acetyl-CoA and glyoxylate (lyase reaction), and in the second cycle it condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA (condensation reaction). The combination of forward and reverse directions of a reversible enzymatic reaction, using two different substrates, is rather uncommon and reduces the number of enzymes required in the pathway. In summary, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase catalyzes the interconversion of L-malyl-CoA plus propionyl-CoA to beta-methylmalyl-CoA plus acetyl-CoA.     

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.     

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.     

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.     

Enzymes of the citramalate cycle in <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis>

Rhodospirillum rubrum is among the bacteria that can assimilate acetate in the absence of isocitrate lyase, the key enzyme of glyoxylate shunt. Previously we have suggested the functioning of a new anaplerotic cycle of acetate assimilation in this bacterium: citramalate cycle, where acetyl-CoA is oxidized to glyoxylate. This work has demonstrated the presence of all the key enzymes of this cycle in R. rubrum extracts: citramalate synthase catalyzing condensation of acetyl-CoA and pyruvate with the formation of citramalate, mesaconase forming mesaconate from L-citramalate, and the enzymes catalyzing transformation of propionyl-CoA + glyoxylate 3-methylmalyl-CoA ? mesaconyl-CoA. At the same time, R. rubrum synthesizes crotonyl-CoA carboxylase/reductase, which is the key enzyme of ethylmalonyl-CoA pathway discovered recently in Rhodobacter sphaeroides. Physiological differences between the citramalate cycle and the ethylmalonyl-CoA pathway are discussed.     

Catabolic and anabolic enzyme activities and energetics of acetone metabolism of the sulfate-reducing bacterium Desulfococcus biacutus.

Acetone degradation by cell suspensions of Desulfococcus biacutus was CO2 dependent, indicating initiation by a carboxylation reaction, while degradation of 3-hydroxybutyrate was not CO2 dependent. Growth on 3-hydroxybutyrate resulted in acetate accumulation in the medium at a ratio of 1 mol of acetate per mol of substrate degraded. In acetone-grown cultures no coenzyme A (CoA) transferase or CoA ligase appeared to be involved in acetone metabolism, and no acetate accumulated in the medium, suggesting that the carboxylation of acetone and activation to acetoacetyl-CoA may occur without the formation of a free intermediate. Catabolism of 3-hydroxybutyrate occurred after activation by CoA transfer from acetyl-CoA, followed by oxidation to acetoacetyl-CoA. In both acetone-grown cells and 3-hydroxybutyrate-grown cells, acetoacetyl-CoA was thioyltically cleaved to two acetyl-CoA residues and further metabolized through the carbon monoxide dehydrogenase pathway. Comparison of the growth yields on acetone and 3-hydroxybutyrate suggested an additional energy requirement in the catabolism of acetone. This is postulated to be the carboxylation reaction (delta G(o)' for the carboxylation of acetone to acetoacetate, +17.1 kJ.mol-1). At the intracellular acyl-CoA concentrations measured, the net free energy change of acetone carboxylation and catabolism to two acetyl-CoA residues would be close to 0 kJ.mol of acetone-1, if one mol of ATP was invested. In the absence of an energy-utilizing step in this catabolic pathway, the predicted intracellular acetoacetyl-CoA concentration would be 10(13) times lower than that measured. Thus, acetone catabolism to two acetyl-CoA residues must be accompanied by the utilization of teh energetic equivalent of (at lease) one ATP molecule. Measurement of enzyme activities suggested that assimilation of acetyl-CoA occurred through a modified citric acid cycle in which isocitrate was cleaved to succinate and glyoxylate. Malate synthase, condensing glyoxylate and acetyl-CoA, acted as an anaplerotic enzyme. Carboxylation of pyruvate of phosphoenolpyruvate could not be detected.     

Degradation of glyoxylate and glycolate with ATP synthesis by a thermophilic anaerobic bacterium, Moorella sp. strain HUC22-1

The thermophilic homoacetogenic bacterium Moorella sp. strain HUC22-1 ferments glyoxylate to acetate roughly according to the reaction 2 glyoxylate --> acetate + 2 CO(2). A batch culture with glyoxylate and yeast extract yielded 11.7 g per mol of cells per substrate, which was much higher than that obtained with H(2) plus CO(2). Crude extracts of glyoxylate-grown cells catalyzed the ADP- and NADP-dependent condensation of glyoxylate and acetyl coenzyme A (acetyl-CoA) to pyruvate and CO(2) and converted pyruvate to acetyl-CoA and CO(2), which are the key reactions of the malyl-CoA pathway. ATP generation was also detected during the key enzyme reactions of this pathway. Furthermore, this bacterium consumed l-malate, an intermediate in the malyl-CoA pathway, and produced acetate. These findings suggest that Moorella sp. strain HUC22-1 can generate ATP by substrate-level phosphorylation during glyoxylate catabolism through the malyl-CoA pathway.     

A reverse glyoxylate shunt to build a non-native route from C4 to C2 in Escherichia coli

Most central metabolic pathways such as glycolysis, fatty acid synthesis, and the TCA cycle have complementary pathways that run in the reverse direction to allow flexible storage and utilization of resources. However, the glyoxylate shunt, which allows for the synthesis of four-carbon TCA cycle intermediates from acetyl-CoA, has not been found to be reversible to date. As a result, glucose can only be converted to acetyl-CoA via the decarboxylation of the three-carbon molecule pyruvate in heterotrophs. A reverse glyoxylate shunt (rGS) could be extended into a pathway that converts C₄ carboxylates into two molecules of acetyl-CoA without loss of CO₂. Here, as a proof of concept, we engineered in Escherichia coli such a pathway to convert malate and succinate to oxaloacetate and two molecules of acetyl-CoA. We introduced ATP-coupled heterologous enzymes at the thermodynamically unfavorable steps to drive the pathway in the desired direction. This synthetic pathway in essence reverses the glyoxylate shunt at the expense of ATP. When integrated with central metabolism, this pathway has the potential to increase the carbon yield of acetate and biofuels from many carbon sources in heterotrophic microorganisms, and could be the basis of novel carbon fixation cycles.     

The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: enzymes of the citramalate cycle in Rhodobacter sphaeroides

The mechanism of acetate assimilation by the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shortcut, has been studied. In a previous work, proceeding from data on acetate assimilation by Rba. sphaeroides cell suspensions, a suggestion was made regarding the operation, in this bacterium, of the citramalate cycle. This cycle was earlier found in Rhodospirillum rubrum in the form of an anaplerotic reaction sequence that operates during growth on acetate instead of the glyoxylate shortcut, which is not present in the latter bacterium. The present work considers the enzymes responsible for acetate assimilation in Rba. sphaeroides. It is shown that this bacterium possesses the key enzymes of the citramalate cycle: citramalate synthase, which catalyzes condensation of acetyl-CoA and pyruvate and, as a result, forms citramalate, and 3-methylmalyl-CoA lyase, which catalyzes the cleavage of 3-methylmalyl-CoA to glyoxylate and propionyl-CoA. The regeneration of pyruvate, which is the acetyl-CoA acceptor in the citramalate cycle, involves propionyl-CoA and occurs via the following reaction sequence: propionyl-CoA (+ CO2) --> methylmalonyl-CoA --> succinyl-CoA --> succinate --> fumarate --> malate --> oxalacetate (- CO2) --> phosphoenolpyruvate --> pyruvate. The independence of the cell growth and the acetate assimilation of CO2 is due to the accumulation of CO2/HCO3- (released during acetate assimilation) in cells to a level sufficient for the effective operation of propionyl-CoA carboxylase.     

Glyoxylate regeneration pathway in the methylotroph Methylobacterium extorquens AM1

Most serine cycle methylotrophic bacteria lack isocitrate lyase and convert acetyl coenzyme A (acetyl-CoA) to glyoxylate via a novel pathway thought to involve butyryl-CoA and propionyl-CoA as intermediates. In this study we have used a genome analysis approach followed by mutation to test a number of genes for involvement in this novel pathway. We show that methylmalonyl-CoA mutase, an R-specific crotonase, isobutyryl-CoA dehydrogenase, and a GTPase are involved in glyoxylate regeneration. We also monitored the fate of (14)C-labeled carbon originating from acetate, butyrate, or bicarbonate in mutants defective in glyoxylate regeneration and identified new potential intermediates in the pathway: ethylmalonyl-CoA, methylsuccinyl-CoA, isobutyryl-CoA, methacrylyl-CoA, and beta-hydroxyisobutyryl-CoA. A new scheme for the pathway is proposed based on these data.     

Characterization of an acyl-CoA: carboxylate CoA-transferase from Aspergillus nidulans involved in propionyl-CoA detoxification

Filamentous fungi metabolize toxic propionyl-CoA via the methylcitrate cycle. Disruption of the methylcitrate synthase gene leads to an accumulation of propionyl-CoA and attenuates virulence of Aspergillus fumigatus . However, addition of acetate, but not ethanol, to propionate-containing medium strongly reduces the accumulation of propionyl-CoA and restores growth of the methylcitrate synthase mutant. Therefore, the existence of a CoA-transferase was postulated, which transfers the CoASH moiety from propionyl-CoA to acetate and, thereby, detoxifying the cell. In this study, we purified the responsible protein from Aspergillus nidulans and characterized its biochemical properties. The enzyme used succinyl-, propionyl- and acetyl-CoA as CoASH donors and the corresponding acids as acceptor molecules. Although the protein displayed high sequence similarity to acetyl-CoA hydrolases this activity was hardly detectable. We additionally identified and deleted the coding DNA sequence of the CoA-transferase. The mutant displayed weak phenotypes in the presence of propionate and behaved like the wild type when no propionate was present. However, when a double-deletion mutant defective in both methylcitrate synthase and CoA-transferase was constructed, the resulting strain was unable to grow on media containing acetate and propionate as sole carbon sources, which confirmed the in vivo activity of the CoA-transferase.     

Influence of nitrate on oxalate- and glyoxylate-dependent growth and acetogenesis by Moorella thermoacetica

Oxalate and glyoxylate supported growth and acetate synthesis by Moorella thermoacetica in the presence of nitrate under basal (without yeast extract) culture conditions. In oxalate cultures, acetate formation occurred concomitant with growth and nitrate was reduced in the stationary phase. Growth in the presence of [(14)C]bicarbonate or [(14)C]oxalate showed that CO(2) reduction to acetate and biomass or oxalate oxidation to CO(2) was not affected by nitrate. However, cells engaged in oxalate-dependent acetogenesis in the presence of nitrate lacked a membranous b-type cytochrome, which was present in cells grown in the absence of nitrate. In glyoxylate cultures, growth was coupled to nitrate reduction and acetate was formed in the stationary phase after nitrate was totally consumed. In the absence of nitrate, glyoxylate-grown cells incorporated less CO(2) into biomass than oxalate-grown cells. CO(2) conversion to biomass by glyoxylate-grown cells decreased when cells were grown in the presence of nitrate. These results suggest that: (1) oxalate-grown cells prefer CO(2) as an electron sink and bypass the nitrate block on the acetyl-CoA pathway at the level of reductant flow and (2) glyoxylate-grown cells prefer nitrate as an electron sink and bypass the nitrate block of the acetyl-CoA pathway by assimilating carbon via an unknown process that supplements or replaces the acetyl-CoA pathway. In this regard, enzymes of known pathways for the assimilation of two-carbon compounds were not detected in glyoxylate- or oxalate-grown cells.     

The ethylmalonyl-CoA pathway is used in place of the glyoxylate cycle by Methylobacterium extorquens AM1 during growth on acetate

Acetyl-CoA assimilation was extensively studied in organisms harboring the glyoxylate cycle. In this study, we analyzed the metabolism of the facultative methylotroph Methylobacterium extorquens AM1, which lacks isocitrate lyase, the key enzyme in the glyoxylate cycle, during growth on acetate. MS/MS-based proteomic analysis revealed that the protein repertoire of M. extorquens AM1 grown on acetate is similar to that of cells grown on methanol and includes enzymes of the ethylmalonyl-CoA (EMC) pathway that were recently shown to operate during growth on methanol. Dynamic 13C labeling experiments indicate the presence of distinct entry points for acetate: the EMC pathway and the TCA cycle. 13C steady-state metabolic flux analysis showed that oxidation of acetyl-CoA occurs predominantly via the TCA cycle and that assimilation occurs via the EMC pathway. Furthermore, acetyl-CoA condenses with the EMC pathway product glyoxylate, resulting in malate formation. The latter, also formed by the TCA cycle, is converted to phosphoglycerate by a reaction sequence that is reversed with respect to the serine cycle. Thus, the results obtained in this study reveal the utilization of common pathways during the growth of M. extorquens AM1 on C1 and C2 compounds, but with a major redirection of flux within the central metabolism. Furthermore, our results indicate that the metabolic flux distribution is highly complex in this model methylotroph during growth on acetate and is fundamentally different from organisms using the glyoxylate cycle.     

The catalytic cycle of biosynthetic thiolase: a conformational journey of an acetyl group through four binding modes and two oxyanion holes

Biosynthetic thiolase catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA. This is a key step in the synthesis of many biological compounds, including steroid hormones and ketone bodies. The thiolase reaction involves two chemically distinct steps; during acyl transfer, an acetyl group is transferred from acetyl-CoA to Cys89, and in the Claisen condensation step, this acetyl group is further transferred to a second molecule of acetyl-CoA, generating acetoacetyl-CoA. Here, new crystallographic data for Zoogloea ramigera biosynthetic thiolase are presented, covering all intermediates of the thiolase catalytic cycle. The high-resolution structures indicate that the acetyl group goes through four conformations while being transferred from acetyl-CoA via the acetylated enzyme to acetoacetyl-CoA. This transfer is catalyzed in a rigid cavity lined by mostly hydrophobic side chains, in addition to the catalytic residues Cys89, His348, and Cys378. The structures highlight the importance of an oxyanion hole formed by a water molecule and His348 in stabilizing the negative charge on the thioester oxygen atom of acetyl-CoA at two different steps of the reaction cycle. Another oxyanion hole, composed of the main chain nitrogen atoms of Cys89 and Gly380, complements a negative charge of the thioester oxygen anion of the acetylated intermediate, stabilizing the tetrahedral transition state of the Claisen condensation step. The reactivity of the active site may be modulated by hydrogen bonding networks extending from the active site toward the back of the molecule.     

Acetate-dependent photoheterotrophic growth and the differential requirement for the Calvin–Benson–Bassham reductive pentose phosphate cycle in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> and <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis>

Rhodobacter sphaeroides ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deletion strain 16 was capable of photoheterotrophic growth with acetate, while Rhodopseudomonas palustris RubisCO-deletion strain 2040 could not grow under these conditions. The reason for this difference lies in the fact that Rba. sphaeroides and Rps. palustris use different pathways for acetate assimilation, the ethylmalonyl-CoA pathway, and glyoxylate-bypass cycle, respectively. The ethylmalonyl-CoA pathway is distinct from the glyoxylate cycle as one molecule of CO₂ and one molecule of HCO₃ ⁻ per three molecules of acetyl-CoA are co-assimilated to form two malate molecules. The glyoxylate cycle directly converts two acetyl-CoA molecules to malate. Each pathway, therefore, also dictates at what point, CO₂ and reductant are consumed, thereby determining the requirement for the Calvin–Benson–Bassham reductive pentose phosphate cycle.     

Mechanism of action of beta-oxoacyl-CoA thiolase from rat liver cytosol. Direct evidence for the order of addition of the two acetyl-CoA molecules during the formation of acetoacetyl-CoA.

Single-turnover enzyme reactions were employed with beta-oxoacyl-CoA thiolase purified from rat liver cytosol to determine the order of binding of the two acetyl-CoA molecules to the enzyme during the formation of acetoacetyl-CoA. Equimolar quantities of [1-14C]acetyl-CoA and enzyme were mixed initially in a rapid mixing device and the reaction was quenched by addition of an excess of unlabelled acetyl-CoA. Degradation of the resulting acetoacetyl-CoA revealed that the larger proportion of the radioactivity was in C-3. In the converse experiment, in which unlabelled acetyl-CoA was mixed with enzyme and the reaction was quenched with [1-14C]acetyl-CoA, radioactivity was incorporated preferentially into C-1. Similar results were obtained when [14C]acetyl-enzyme complex isolated by gel filtration was reacted with unlabelled acetyl-CoA, the radioactivity appearing largely in C-3. These findings lead to the conclusion that of the two molecules of acetyl-CoA that are bound by the enzyme and converted into acetoacetyl-CoA, it is the one giving rise to C-3 and -4 that is bound initially to the enzyme in the form of the acetyl-enzyme intermediate complex.     

The methylaspartate cycle in haloarchaea and its possible role in carbon metabolism

Haloarchaea (class Halobacteria) live in extremely halophilic conditions and evolved many unique metabolic features, which help them to adapt to their environment. The methylaspartate cycle, an anaplerotic acetate assimilation pathway recently proposed for Haloarcula marismortui, is one of these special adaptations. In this cycle, acetyl-CoA is oxidized to glyoxylate via methylaspartate as a characteristic intermediate. The following glyoxylate condensation with another molecule of acetyl-CoA yields malate, a starting substrate for anabolism. The proposal of the functioning of the cycle was based mainly on in vitro data, leaving several open questions concerning the enzymology involved and the occurrence of the cycle in halophilic archaea. Using gene deletion mutants of H. hispanica, enzyme assays and metabolite analysis, we now close these gaps by unambiguous identification of the genes encoding all characteristic enzymes of the cycle. Based on these results, we were able to perform a solid study of the distribution of the methylaspartate cycle and the alternative acetate assimilation strategy, the glyoxylate cycle, among haloarchaea. We found that both of these cycles are evenly distributed in haloarchaea. Interestingly, 83% of the species using the methylaspartate cycle possess also the genes for polyhydroxyalkanoate biosynthesis, whereas only 34% of the species with the glyoxylate cycle are capable to synthesize this storage compound. This finding suggests that the methylaspartate cycle is shaped for polyhydroxyalkanoate utilization during carbon starvation, whereas the glyoxylate cycle is probably adapted for growth on substrates metabolized via acetyl-CoA.     

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.     

Dark metabolism of acetate in Rhodospirillum rubrum cells, grown under photoheterotropic conditions

The mechanism of the dark assimilation of acetate in the photoheterotrophically grown nonsulfur bacterium Rhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation in Rsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle in Rsp. rubrum cells grown aerobically in the dark can function as an anaplerotic pathway. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicative that citramalate and mesaconate are intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts of Rsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function in Rsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.