Microbial metabolism of aliphatic glycols. Bacterial metabolism of ethylene glycol

A species of Flavobacterium isolated from pond water by its ability to grow aerobically on ethylene glycol as the role source of carbon initially oxidised the diol to glyoxylate via glycollate. The glyoxylate was metabolised by the glycerate pathway to acetyl-CoA. The acetyl-CoA was further metabolised by the tricarboxylic acid cycle plus malate synthase acting anaplerotically.     

Bacterial metabolism of ethylene glycol

Metabolism of ethylene glycol as the sole source of carbon by a species of Flavobacterium was affected by the dissolved oxygen tension of the growth medium. Under strongly aerobic conditions the diol was exclusively metabolised to glycollate by an initial oxidase, subsequently metabolised to acetyl-CoA with no net change in ATP, and then oxidised to CO₂, by the tricarboxylic acid cycle yielding large amounts of reduced nicotinamide nucleotides which were used to generate a net gain in ATP by oxidative phospsorylation. Under miccroaerophilic conditions, some ethylene glycol after initial metabolism to acetyl-CoA by the oxidase-initiated pathway, was subsequently catabolised to acetyl phosphate and then acetate, yielding a net gain in ATP by substrate-level phosphorylation: additionally some diol was catabolised by an inducible diol dehydratase to acetaldehyde and subsequently reduced to ethanol as a terminal metabolite.     

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.     

The ethanolic fermentation pathway supports respiration and lipid biosynthesis in tobacco pollen

Rapid pollen tube growth requires a high rate of sugar metabolism to meet energetic and biosynthetic demands. Previous work on pollen sugar metabolism showed that tobacco pollen carry out efficient ethanolic fermentation concomitantly with a high rate of respiration (Bucher et al., 1995). Here we show that the products of fermentation, acetaldehyde and ethanol, are further metabolised in a pathway that bypasses mitochondrial PDH. The enzymes involved in this pathway are pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthetase. Radiolabelling experiments show that during tobacco pollen tube growth label of 14C-ethanol is incorporated into CO2 as well as into lipids and other higher molecular weight compounds. A role for the glyoxylate cycle appears unlikely since activity of malate synthase, a key enzyme of the glyoxylate cycle, could not be detected.     

Metabolism of threonine by Fusaria growth on threonine as the sole carbon and nitrogen source

Three strains ofFusarium supporting aerobic growth onl-threonine as the sole source of energy and carbon and nitrogen, initially metabolised threonine to acetyl-CoA and glycine via induciblel-threonine:NAD⁺ dehydrogenase plus 2-amino-3-oxobutyrate:CoA ligase activities. Comparative enzyme induction patterns after growth of the three strains on a wide range of carbon sources indicated that the glycine produced by the NAD⁺ plus CoASH-dependent cleavage of threonine was subsequently utilised as an energy source and biosynthetic precursor via the glycine-serine pathway, pyruvate carboxylase, and ultimately the TCA cycle. Acetyl-CoA, the second initial C₂ threonine catabolism product, was subsequently assimilated via a combined TCA plus glyoxylate cycle.     

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.     

Link between primary and secondary metabolism in the biotransformation of trimethylammonium compounds by escherichia coli

The aim of this work was to understand the steps controlling the process of biotransformation of trimethylamonium compounds into L(-)-carnitine by Escherichia coli and the link between the central carbon or primary and the secondary metabolism expressed. Thus, the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration reaction and crotonobetaine reduction reaction), in the synthesis of acetyl-CoA (pyruvate dehydrogenase, acetyl-CoA synthetase, and ATP:acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (isocitrate dehydrogenase) and glyoxylate (isocitrate lyase) cycles, were followed in batch with both growing and resting cells and during continuous cell growth in stirred-tank and high-cell-density membrane reactors. In addition, the levels of carnitine, crotonobetaine, gamma-butyrobetaine, ATP, NADH/NAD(+), and acetyl-CoA/CoA ratios were measured to determine how metabolic fluxes were distributed in the catabolic system. The results provide the first experimental evidence demonstrating the important role of the glyoxylate shunt during biotransformation of resting cells and the need for high levels of ATP to maintain metabolite transport and biotransformation (2.1 to 16.0 mmol L cellular/mmol ATP L reactor h). Moreover, the results obtained for the pool of acetyl-CoA/CoA indicate that it also correlated with the biotransformation process. The main metabolic pathway operating during cell growth in the high cell-density membrane reactor was that related to isocitrate dehydrogenase (during start-up) and isocitrate lyase (during steady-state operation), together with phosphotransacetylase and acetyl-CoA synthetase. More importantly, the link between central carbon and L(-)-carnitine metabolism at the level of the ATP pool was also confirmed.     

Microbial metabolism of ethylene

The ethylene-oxidizing strain E20 was grown on different carbon sources to obtain information on the metabolism of ethylene from simultaneous adaptation studies and from measurements of specific activities of enzymes in cell-free extracts.From the simultaneous adaptation studies it was concluded that ethylene oxide is a product of ethylene catabolism. The bacterium was also able to grow on the epoxide. From a comparison of the specific activities of isocitrate lyase and malate synthetase in different extracts it was concluded that the glyoxylate cycle was involved in the metabolism of ethylene, indicating that acetyl-CoA is a metabolite of ethylene catabolism. The sequence of reactions leading from ethylene oxide to acetyl-CoA could not be established from the simultaneous adaptation experiments and the enzyme activities in extracts.Support for the research has come in part from grants of the N.V. Nederlandse Gasunie and the VEG Gasinstituut.     

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.     

Growth of Penicillium janthinellum on glycine as sole carbon and nitrogen source

Penicillium janthinellum is able to grow on glycine as the sole carbon and nitrogen source. The amino acid is transaminated to glyoxylate which is further metabolised to pyruvate by the glycerate pathway. The reaction product of partially purified glycerate kinase from this fungus is 2-phosphoglycerate. Phosphoglycerate mutase initiates gluconeogenesis from glycine. Partially purified phosphoglycerate mutase is inhibited by fructose 6-phosphate. The possible significance of this regulation is discussed.     

Fermentative degradation of glyoxylate by a new strictly anaerobic bacterium

A new strictly anaerobic, gram-negative, nonsporeforming bacterium, Strain PerGlx1, was enriched and isolated from marine sediment samples with glyoxylate as sole carbon and energy source. The guanineplus-cytosine content of the DNA was 44.1±0.2 mol %. Glyoxylate was utilized as the only substrate and was stoichiometrically degraded to carbon dioxide, hydrogen, and glycolate. An acetyl-CoA and ADP-dependent glyoxylate converting enzyme activity, malic enzyme, and pyruvate synthase were found at activities sufficient for growth (0.25 U x mg protein^-1). These findings allow to design a new degradation pathway for glyoxylate: glyoxylate is condensed with acetyl-CoA to form malyl-CoA; the free energy of the thioester linkage in malyl-CoA is conserved by substrate level phosphorylation. Part of the electrons released during glyoxylate oxidation to CO₂ reduce a small fraction of glyoxylate to glycolate.     

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.     

Cardiolipin-deficient cells depend on anaplerotic pathways to ameliorate defective TCA cycle function

Previous studies have shown that the cardiolipin (CL)-deficient yeast mutant, crd1Δ, has decreased levels of acetyl-CoA and decreased activities of the TCA cycle enzymes aconitase and succinate dehydrogenase. These biochemical phenotypes are expected to lead to defective TCA cycle function. In this study, we report that signaling and anaplerotic metabolic pathways that supplement defects in the TCA cycle are essential in crd1Δ mutant cells. The crd1Δ mutant is synthetically lethal with mutants in the TCA cycle, retrograde (RTG) pathway, glyoxylate cycle, and pyruvate carboxylase 1. Glutamate levels were decreased, and the mutant exhibited glutamate auxotrophy. Glyoxylate cycle genes were up-regulated, and the levels of glyoxylate metabolites succinate and citrate were increased in crd1Δ. Import of acetyl-CoA from the cytosol into mitochondria is essential in crd1Δ, as deletion of the carnitine-acetylcarnitine translocase led to lethality in the CL mutant. β-oxidation was functional in the mutant, and oleate supplementation rescued growth defects. These findings suggest that TCA cycle deficiency caused by the absence of CL necessitates activation of anaplerotic pathways to replenish acetyl-CoA and TCA cycle intermediates. Implications for Barth syndrome, a genetic disorder of CL metabolism, are discussed.     

The Apparent Malate Synthase Activity of Rhodobacter sphaeroides Is Due to Two Paralogous Enzymes, (3S)-Malyl-Coenzyme A (CoA)/β-Methylmalyl-CoA Lyase and (3S)- Malyl-CoA Thioesterase

Assimilation of acetyl coenzyme A (acetyl-CoA) is an essential process in many bacteria that proceeds via the glyoxylate cycle or the ethylmalonyl-CoA pathway. In both assimilation strategies, one of the final products is malate that is formed by the condensation of acetyl-CoA with glyoxylate. In the glyoxylate cycle this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway the reaction is separated into two proteins: malyl-CoA lyase, a well-known enzyme catalyzing the Claisen condensation of acetyl-CoA with glyoxylate and yielding malyl-CoA, and an unidentified malyl-CoA thioesterase that hydrolyzes malyl-CoA into malate and CoA. In this study the roles of Mcl1 and Mcl2, two malyl-CoA lyase homologs in Rhodobacter sphaeroides, were investigated by gene inactivation and biochemical studies. Mcl1 is a true (3S)-malyl-CoA lyase operating in the ethylmalonyl-CoA pathway. Notably, Mcl1 is a promiscuous enzyme and catalyzes not only the condensation of acetyl-CoA and glyoxylate but also the cleavage of β-methylmalyl-CoA into glyoxylate and propionyl-CoA during acetyl-CoA assimilation. In contrast, Mcl2 was shown to be the sought (3S)-malyl-CoA thioesterase in the ethylmalonyl-CoA pathway, which specifically hydrolyzes (3S)-malyl-CoA but does not use β-methylmalyl-CoA or catalyze a lyase or condensation reaction. The identification of Mcl2 as thioesterase extends the enzyme functions of malyl-CoA lyase homologs that have been known only as “Claisen condensation” enzymes so far. Mcl1 and Mcl2 are both related to malate synthase, an enzyme which catalyzes both a Claisen condensation and thioester hydrolysis reaction.Many organic compounds are initially metabolized to acetyl coenzyme A (acetyl-CoA), at which point they enter the central carbon metabolism. Examples of such growth substrates are C₁ and C₂ compounds (e.g., methanol and ethanol), fatty acids, waxes, esters, alkenes, or (poly)hydroxyalkanoates. The synthesis of all cell constituents from acetyl-CoA requires a specialized pathway for the conversion of this central C₂ unit into other biosynthetic precursor metabolites. This (anaplerotic) process is referred to as acetyl-CoA assimilation, and two very different strategies have been described, i.e., the glyoxylate cycle and the ethylmalonyl-CoA pathway (12, 21) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Pathways for acetyl-CoA assimilation. (A) Glyoxylate cycle. The key enzymes are isocitrate lyase and malate synthase. (B) Ethylmalonyl-CoA pathway. The unique enzymes of the pathway are crotonyl-CoA carboxylase/reductase, ethylmalonyl-CoA/methylmalonyl-CoA epimerase, (2R)-ethylmalonyl-CoA mutase, (2S)-methylsuccinyl-CoA dehydrogenase, mesaconyl-CoA hydratase, (3S)-malyl-CoA/β-methylmalonyl-CoA lyase, and (3S)-malyl-CoA thioesterase. The enzymes involved in the (apparent) malate synthase reaction(s) are boxed for each pathway.The glyoxylate cycle for acetyl-CoA assimilation is in fact a modified citric acid cycle that converts two molecules of acetyl-CoA to the citric acid cycle intermediate malate (Fig. ​(Fig.1A)1A) (21). In a first reaction sequence, one molecule of acetyl-CoA is converted into glyoxylate due to the combined action of the initial enzymes of the citric acid cycle and isocitrate lyase, the key enzyme of this assimilation strategy. Isocitrate lyase cleaves the citric cycle intermediate isocitrate into succinate and glyoxylate (22). The glyoxylate formed is then condensed in a second step with another molecule of acetyl-CoA to yield malate and free CoA. Because the two decarboxylation reactions of the citric acid cycle are circumvented by this acetyl-CoA assimilation strategy, the glyoxylate cycle is also referred to as the “glyoxylate bypass” or “glyoxylate shunt.”The ethylmalonyl-CoA pathway for acetyl-CoA assimilation replaces the glyoxylate cycle in bacteria that lack isocitrate lyase (1, 12). In this linear pathway, three molecules of acetyl-CoA, one molecule of CO₂, and one molecule of bicarbonate are converted to the citric acid cycle intermediates succinyl-CoA and malate (Fig. ​(Fig.1B).1B). The ethylmalonyl-CoA pathway requires at least seven unique enzymes. Crotonyl-CoA carboxylase/reductase, ethylmalonyl-CoA mutase, and methylsuccinyl-CoA dehydrogenase are considered key enzymes of the pathway, and all three enzymes have been characterized from Rhodobacter sphaeroides (12-14).Although these two acetyl-CoA strategies differ with respect to their reaction sequence, intermediates and overall balance, the glyoxylate cycle and the ethylmalonyl-CoA pathway both require the condensation of acetyl-CoA and glyoxylate to form malate (Fig. ​(Fig.1,1, boxed). In the glyoxylate cycle, this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway malate synthase is catalyzed by two separate enzymes, malyl-CoA lyase and malyl-CoA thioesterase (7, 26).Malyl-CoA lyases catalyze the reversible condensation of acetyl-CoA and glyoxylate into malyl-CoA and have been purified from Methylobacterium extorquens, Chloroflexus aurantiacus, Aminobacter aminovorans, and Rhodobacter capsulatus; the corresponding genes were identified as mclA (M. extorquens), mcl (C. aurantiacus), and mcl1 (R. capsulatus) (5, 16, 17, 19, 26). Remarkably, these proteins are promiscuous enzymes that also catalyze the (reversible) cleavage of β-methylmalyl-CoA into glyoxylate and propionyl-CoA, and it has been suggested that these enzymes catalyze both reactions in vivo (16, 19, 26). However, in contrast to malyl-CoA lyase, the malyl-CoA thioesterase catalyzing the highly exergonic hydrolysis of the CoA-thioester into malate and free CoA has not been identified so far, and the nature of the enzyme has remained enigmatic (7, 26).For R. sphaeroides, a malyl-CoA lyase homolog has been shown to be upregulated during growth on acetate, and it was proposed that this protein (Mcl1) catalyzes the cleavage of β-methylmalyl-CoA, as well as the condensation of acetyl-CoA and glyoxylate in the ethylmalonyl-CoA pathway (1). Interestingly, R. sphaeroides encodes a second malyl-CoA lyase homolog with 34% amino acid sequence identity to Mcl1. This protein, named Mcl2, was also shown to be upregulated during growth of R. sphaeroides on acetate, but a function could not be assigned so far (1). We therefore addressed the function of both malyl-CoA lyase homologs by gene inactivation and biochemical studies of recombinant Mcl1 and Mcl2. Based on our findings, we confirm here the function of Mcl1 in R. sphaeroides as (3S)-malyl-CoA/β-methylmalyl-CoA lyase and identify its paralog Mcl2 as the long-sought (3S)-malyl-CoA thioesterase.     

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.     

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.     

Generation of glyoxylate in methylotrophic bacteria

The mode of glyoxylate production from acetyl-CoA was investigated in three strains of methylotrophic bacteria,Pseudomonas MA,Pseudomonas AM1 and organism PAR. This investigation was prompted by the recently reported discovery of a homoisocitrate lyase in methylotrophic bacteria and the suggested involvement of this novel enzyme in assimilation of C₁ and C₂ compounds as part of a homoisocitrate-glyoxylate cycle. We were unable to detect cleavage of any of the four stereoisomers of homoisocitric acid by cell-free extracts of C₁-or C₂-grown bacteria. Extracts of C₁-grown bacteria did not catalyze condensation of glyoxylate with glutarate or production of glyoxylate from acetyl-CoA and 2-ketoglutarate. Extracts of C₁-grownPseudomonas MA catalyzed cleavage of isocitrate;threo-homoisocitrate was a potent competitive inhibitor of this reaction. These results indicate that homoisocitrate cleavage does not occur in any of the methylotrophs tested. The pathway for oxidation of acetyl-CoA to glyoxylate inPseudomonas AM1 and organism PAR therefore remains obscure.     

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.