3-Deoxy-D-pentulosonic acid aldolase and its role in a new pathway of D-xylose degradation

Evidence is presented for a new pathway of D-xylose metabolism. Cell-free extracts prepared from a pseudomonad grown on D-xylose as a sole carbon source contain prominent D-xylose dehydrogenase, D-xylo-aldonate dehydrase and a new aldolase which cleaves 2-keto-3-deoxy-D-xylonate (3-deoxy-D-pentulosonic acid) to pyruvate and glycolaldehyde. The aldolase is specific for the D-isomer and is distinguishable from a previously described enzyme present in the same organism which is correspondingly specific for the L-isomer. The subsequent conversion of 3-deoxy-D-pentulosonic acid to α-keto-glutarate which has been reported in other organisms could not be demonstrated. Thus, the pathway of D-xylose degradation in this pseudomonad is believed to be: D-xylose → D-xylonate → 3-deoxy-D-pentulosonic acid → pyruvate + glycolaldehyde.     

The microbial metabolism of acetophenone. Metabolism of acetophenone and some chloroacetophenones by an Arthrobacter species

1. An organism that utilizes acetophenone as sole source of carbon and energy was isolated in pure culture and tentatively identified as an Arthrobacter sp. 2. Cell-free extracts of the acetophenone-grown organism contained an enzyme, acetophenone oxygenase, that catalysed an NADPH-dependent consumption of O(2) in the presence of the growth substrate; approx. 1mol of O(2) and 1mol of NADPH were consumed per mol of acetophenone oxidized. 3. Cell-free extracts also contained an enzyme capable of the hydrolysis of phenyl acetate to phenol and acetate. The amount of this esterase was increased markedly by growth on acetophenone. 4. The observed products of the acetophenone oxygenase reaction by crude cell-free extracts were phenol and acetate. However, inhibition of the phenyl acetate esterase by paraoxon resulted in the formation of phenyl acetate from acetophenone. 5. A degradative sequence is proposed in which acetophenone is metabolized by an oxygen-insertion reaction to form phenyl acetate. Further metabolism occurs by hydrolysis of this ester. 6. The organism and extracts were shown to metabolize chlorinated acetophenones. The environmental implications of this observation are discussed.     

Pathway of degradation of nitrilotriacetate by a Pseudomonas species.

The pathway of degradation of nitrilotriacetate (NTA) was determined by using cell-free extracts and a 35-fold purification of NTA monooxygenase. The first step in the breakdown was an oxidative cleavage of the tertiary amine by the monooxygenase to form the aldo acid, glyoxylate, and the secondary amine, iminodiacetate (IDA). NTA N-oxide acted as a substrate analog for induction of the monooxygenase and was slowly metabolized by the enzyme, but was not an intermediate in the pathway. No intermediate before IDA was found, but an unstable alpha-hydroxy-NTA intermediate was postulated. IDA did undergo cleavage in the presence of the purified monooxygenase to give glyoxylate and glycine, but was not metabolized in cell-free extracts. Glyoxylate was further metabolized by cell-free extracts to yield CO2 and glycerate or glycine, products also found from NTA metabolism. Of the three bacterial isolates in which the NTA pathway has been studied, two strains, one isolated from a British soil and ours from a Michigan soil, appear to be almost identical.     

Acetate utilization by a methylotrophic bacterium

The mode of acetate ultilization in the Gram-negative diplococcoid bacterium PAR was investigated. This organism uses the isocitrate lyase-negative serine pathway during growth on C₁-compounds and was found to lack isocitrate lyase on acetate growth also. Enzyme assays revealed the absence of the glycerate and hydroxyaspartate pathways for the metabolism of C₂-compounds. Pulse-labeling experiments indicated the rapid formation of glycine, glutamate, and malate. The rapid formation of malate and the presence of malate synthase suggest that glyoxylate is an intermediate formed from acetate by a means other than the conventional isocitrate cleavage reaction.     

Bacterial catabolism of threonine. Threonine degradation initiated by L-threonine-NAD+ oxidoreductase.

1. Isolates representing seven bacterial genera capable of growth on L-threonine medium, and possessing high L-threonine 3-dehydrogenase activity, were examined to elucidate the catabolic route. 2. The results of growth, manometric and enzymic experiments indicated the catabolism of L-threonine by cleavage to acetyl-CoA plus glycine, the glycine being further metabolized via L-serine to pyruvate, in all cases. No evidence was obtained of a role for aminoacetone in threonine catabolism or for the metabolism of glycine by the glycerate pathway. 3. The properties of a number of key enzymes in L-threonine catabolism were investigated. The inducibly formed L-threonine 3-dehydrogenase, purified from Corynebacterium sp. B6 to a specific activity of about 30-35 mumol of product formed/min per mg of protein, exhibited a sigmoid kinetic response to substrate concentration. The half-saturating concentration of substrate, [S]0.5, was 20mM and the Hill constant (h) was 1.50. The Km for NAD+ was 0.8mM. The properties of the enzyme were studied in cell-free extracts of other bacteria. 4. New assays for 2-amino-3-oxobutyrate-CoA ligase were devised. The Km for CoA was determined for the first time and found to be 0.14mM at pH8, for the enzyme from Corynebacterium sp. B6. Evidence was obtained for the efficient linkage of the dehydrogenase and ligase enzymes. Cell-free extracts all possessed high activities of the inducibly formed ligase. 5. L-Serine hydroxymethyltransferase was formed constitutively by all isolates, whereas formation of the 'glycine-cleavage system' was generally induced by growth on L-threonine or glycine. The coenzyme requirements of both enzymes were established, and their linked activity in the production of L-serine from glycine was demonstrated by using extracts of Corynebacterium sp. B6. 6. L-Serine dehydratase, purified from Corynebacterium sp. B6 to a specific activity of about 4mumol of product formed/min per mg of protein, was found to exhibit sigmoid kinetics with an [S]0.5 of about 20mM and h identical to 1.4. Similar results were obtained with enzyme preparations from all isolates. The enzyme required Mg2+ for maximum activity, was different from the L-threonine dehydratase also detectable in extracts, and was induced by growth on L-threonine or glycine.     

The plant-like C2 glycolate cycle and the bacterial-like glycerate pathway cooperate in phosphoglycolate metabolism in cyanobacteria

The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO(2)-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO(2). Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO(2). These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO(2), glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

Mutation modifying the serine pathway in methylotrophic bacteria

Methylotrophic bacteria, Gram-positive, with the serine pathway, were shown to have their growth inhibited by 0.5 % glycine. The effects of this amino acid on individual enzyme activities were studied in wild and mutant strains ofMicrococcus varians andBacillus licheniformis. The enzymes studied were glycerate dehydrogenase (EC 1.1.1.29), isocitrate lyase (EC 4.1.3.1), serine hydroxymethyltransferase (EC 2.1.2.1) and glycine—oxaloacetate aminotransferase (EC 2.6.1.35). The last-named enzyme was found to be inhibited, the kinetic constants having been determined for two strain types.     

Eubacterium acidaminophilum sp. nov., a versatile amino acid-degrading anaerobe producing or utilizing H2 or formate

An obligately anaerobic, rod-shaped bacterium was isolated on alanine in co-culture with H₂-scavenging Desulfovibrio and obtained in pure culture with glycine as sole fermentation substrate. The isolated strain, al-2, was motile by a polar to subpolar flagellum and stained Gram-positive. The guanine plus cytosine content of the DNA was 44.0 mol%. Strain al-2 grew in defined, reduced glycine media supplemented with biotin. The pure culture fermented 4 mol glycine to 3 mol acetate, 4 mol ammonia and 2 mol CO₂. Under optimum conditions (34°C, pH 7.3), the doubling time on glycine was 60 min and the molar growth yield 7.6 g cell dry mass. Serine was fermented to acetate, ethanol, CO₂, H₂ and ammonia. In addition, betaine, sarcosine or creatine served as substrates for growth and acetate production if H₂, formate or e.g. valine were added as H-donors. In pure culture on alanine under N₂, strain al-2 grew very poorly and produced H₂ up to a partial pressure of 3.6 kPa (0.035 atm). Desulfovibrio species, Methanospirillum hungatei and Acetobacterium woodii served as H₂-scavengers that allowed good syntrophic growth on alanine. The co-cultures also grew on aspartate, leucine, valine or malate. Alanine and aspartate were stoichiometrically degraded to acetate and ammonia, whereas the reducing equivalents were recovered as H₂S, CH₄ or newly synthetized acetate, respectively. Growth of strain al-2 in co-culture with the hydrogenase-negative, formate-utilizing Desulfovibrio baarsii indicated that a syntrophy was also possible by interspecies formate transfer. Growth on glycine, or on betaine, sarcosine or creatine (plus H-donors) depended strictly on the addition of selenite (0.1 M); selenite was not required for fermentation of serine, or for degradation of alanine, aspartate or valine by the co-cultures. Cell-free extracts of glycine-grown cells contained active glycine reductase, glycine decarboxylase and reversible methyl viologen-dependent formate dehydrogenase in addition to the other enzymes necessary for an oxidation to CO₂. In all reactions NADP was the preferred H-carrier. Both formate and glycine could be synthesized from bicarbonate. Serine-grown cells did not contain serine hydroxymethyl transferase but serine dehydratase and other enzymes commonly involved in pyruvate metabolism to acetate, CO₂ and H₂. The enzymes involved in glycine metabolism were repressed during growth on serine. By its morphology and physiology, strain al-2 did not resemble described amino acid-degrading species. Therefore, the new isolate is proposed as type strain of a new species, Eubacterium acidaminophilum.     

The pathway of glycollate utilization in Chlorella pyrenoidosa

1. Exogenous glycollate was rapidly metabolized in both the light and the dark by photoautotrophically grown Chlorella pyrenoidosa. 2. The incorporation of (14)C from [1-(14)C]glycollate by these cells was inhibited by the tricarboxylic acid-cycle inhibitors monofluoroacetate, diethylmalonate and arsenite, and also by alpha-hydroxypyrid-2-ylmethanesulphonate and isonicotinylhydrazine. 3. Short-term kinetic experiments showed over 80% of the total (14)C present in the soluble fraction from the cells to be in glycine and serine after 10s. This percentage decreased with time whereas the percentage radioactivity in glycerate increased for up to 30s then remained steady. The percentage of the total radioactivity present in citrate increased over the experimental period. Malate was the only other tricarboxylic acid-cycle intermediate to become labelled. 4. The kinetic and inhibitor experiments supported the following pathway of glycollate incorporation: glycollate --> glyoxylate --> glycine --> serine --> hydroxypyruvate --> glycerate --> 3-phosphoglycerate --> 2-phosphoglycerate --> phosphoenolpyruvate --> pyruvate --> acetyl-CoA. 5. The specific activities of the enzymes catalysing this metabolic sequence in cell-free extracts were great enough to account for the observed rate of glycollate metabolism of 0.25mumol/h per mg dry wt. of cells in the light.     

Microbial metabolism of C1 and C2 compounds. The role of glyoxylate, glycollate and acetate in the growth of Pseudomonas AM1 on ethanol and on C1 compounds

Succinate (or a product of succinate metabolism) is a catabolite repressor of some enzymes of the serine pathway (hydroxypyruvate reductase, serine-glyoxylate aminotransferase and glycerate kinase) but not of methanol dehydrogenase nor methylamine dehydrogenase. A mutant (PCT64) of Pseudomonas AM1, which is unable to grow on C(1) compounds, lacks glycerate kinase, showing that this enzyme is essential for the operation of the serine pathway. Mutant PCT48, unable to convert acetate into glycollate, has lost the ability to grow both on C(1) compounds and on ethanol. The properties of a third mutant (PCT57) show that Pseudomonas AM1 contains enzymes catalysing the conversion of acetate into glyoxylate. Evidence is presented that hydroxypyruvate reductase is involved in the oxidation of glycollate to glyoxylate during growth on ethanol. A scheme is proposed for the conversion of ethanol and of C(1) compounds into glyoxylate in which acetate (or a derivative) and glycollate are intermediates.     

Microbial metabolism of aromatic nitriles. Enzymology of C–N cleavage by Nocardia sp. (Rhodochrous group) N.C.I.B. 11216

1. An organism utilizing benzonitrile as sole carbon and nitrogen source was isolated by the enrichment-culture technique and identified as a Nocardia sp. of the rhodochrous group. 2. Respiration studies indicate that nitrile degradation proceeds through benzoic acid and catechol. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme that catalyses the conversion of benzonitrile directly into benzoic acid without intermediate formation of benzamide. 4. This nitrilase enzyme was purified by DEAE-cellulose chromatography and gel filtration on Sephadex G-100 in the presence and absence of substrate. The purity of the enzyme was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme shows a time-dependent substrate-activation process in which the substrate catalyses the association of inactive subunits of mol.wt. 45000 to form the polymeric 12-unit active enzyme of mol.wt. 560000. The time required for complete association is highly dependent on the concentration of the enzyme, temperature and pH. 6. The associated enzyme has a pH optimum of 8.0 and K(m) with benzonitrile as substrate of 4mm. The activation energy of the reaction as deduced from the Arrhenius plot is 51.8kJ/mol. 7. Enzyme activity is inhibited by thiol-specific reagents and several metal ions. 8. Studies with different substrates indicate that the nitrilase is specific for nitrile groups directly attached to the benzene ring. Various substituents in the ring are compatible with activity, though ortho-substitution, except by fluorine, renders the nitrile invulnerable to attack. 9. The environmental implications of these findings and the possible significance of the enzyme in the regulation of metabolism are discussed.     

Effect of adenosine 3',5'-monophosphate on serine metabolism in chick tissues.

The effect of cyclic 3',5'-AMP and supplemental dietary glycine upon de novo synthesis of serine metabolic enzymes in chick livers were examined. Chicks fed crystalline amino acid diets containing 2% glycine had approximately twofold the activity in liver for 3-phosphoglycerate dehydrogenase and phosphoserine phosphatase compared to liver tissue from chicks fed diets lacking in dietary glycine. Chicks subjected to daily intraperitoneal injections of cyclic 3',5'-AMP and fed diets containing no dietary glycine contained biosynthetic enzyme activity similar to glycine-fed chicks suggesting a correlation between glycine and cyclic AMP for serine enzyme induction. The elevated enzyme activity in liver of chicks fed dietary glycine or injected with cyclic AMP was inhibited when chicks were also injected with actinomycin D indicating de novo synthesis of 3-phosphoglycerate dehydrogenase and phosphoserine phosphatase. Dietary glycine or cyclic AMP, however, did not change serine dehydratase and glycerate dehydrogenase activities in chick liver.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

The biosynthesis of serine and glycine in Pseudomonas AM1 with special reference to growth on carbon sources other than C1 compounds

1. A mutant, 20S, of Pseudomonas AM1 was obtained that requires a supplement of serine to grow on succinate, lactate or ethanol. This mutant lacks phosphoserine phosphatase and revertants to wild-type phenotype regained this enzymic activity showing that the phosphorylated pathway of serine biosynthesis is necessary for growth on these three substrates. 2. The requirement for supplemental serine by mutant 20S could be met by glycine, suggesting that Pseudomonas AM1 can obtain C(1) units from glycine. 3. Mutant 20S grows on C(1) compounds at a lower rate compared with the wild type. Supplementation with serine stimulated the growth rate of the mutant suggesting that the phosphorylated pathway of serine biosynthesis plays some role, but not an essential role, during growth on C(1) compounds. 4. A mutant, 82G, was obtained that requires a supplement of glycine to grow on succinate, lactate or ethanol. When grown in such supplemented media, the mutant lacks serine hydroxymethyltransferase and revertants to wild-type phenotype regained enzymic activity showing that during growth on succinate, lactate or ethanol, glycine is made from serine via serine hydroxymethyltransferase, and that the organism can obtain C(1) units from glycine. 5. Mutant 82G grew on methanol and then contained serine hydroxymethyltransferase suggesting that this enzyme is necessary for growth on C(1) compounds and that Pseudomonas AM1 may synthesize two such enzymes, one used in growth on C(1) compounds, the other used in growth on other substrates. Mutant 82G might lack the latter enzyme. 6. Phosphoglycerate dehydrogenase is specifically inhibited by l-serine and the regulatory implications of this are discussed.     

Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L.

A comparative study is presented of the activities of enzymes of glycine and serine metabolism in leaves, germinated cotyledons and root apices of pea (Pisum sativum L.). Data are given for aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate, for enzymes associated with serine synthesis from 3-phosphoglycerate and for glycine decarboxylase and serine hydroxymethyltransferase. Aminotransferase activities differ between the tissues in that, firstly, appreciable transamination of serine, hydroxypyruvate and asparagine occurs only in leaf extracts and, secondly, glyoxylate is transaminated more actively than pyruvate in leaf extracts, whereas the converse is true of extracts of cotyledons and root apices. Alanine is the most active amino-group donor to both glyoxylate and hydroxypyruvate. 3-Phosphoglycerate dehydrogenase and glutamate: O-phosphohydroxypyruvate aminotransferase have comparable activities in all three tissues, except germinated cotyledons, in which the aminotransferase appears to be undetectable. Glycollate oxidase is virtually undetectable in the non-photosynthetic tissues and in these tissues the activity of glycerate dehydrogenase is much lower than that of 3-phosphoglycerate dehydrogenase. Glycine decarboxylase activity in leaves, measured in the presence of oxaloacetate, is equal to about 30–40% of the measured rate of CO₂ fixation and is therefore adequate to account for the expected rate of photorespiration. The activity of glycine decarboxylase in the non-photosynthetic tissues is calculated to be about 2–5% of the activity in leaves and has the characteristics of a pyridoxal-and tetrahydrofolate-dependent mitochondrial reaction; it is stimulated by oxaloacetate, although not by ADP. In leaves, the measured activity of serine hydroxymethyltransferase is somewhat lower than that of glycine decarboxylase, whereas in root apices it is substantially higher. Differential centrifugation of extracts of root apices suggests that an appreciable proportion of serine hydroxymethyltransferase activity is associated with the plastids.Abbreviation GOGAT l-Glutamine:2-oxoglutarate aminotransferase     

Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.     

The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end

Hydroxypyruvate (HP) is an intermediate of the photorespiratory pathway that originates in the oxygenase activity of the key enzyme of photosynthetic CO(2) assimilation, Rubisco. In course of this high-throughput pathway, a peroxisomal transamination reaction converts serine to HP, most of which is subsequently reduced to glycerate by the NADH-dependent peroxisomal enzyme HP reductase (HPR1). In addition, a NADPH-dependent cytosolic HPR2 provides an efficient extraperoxisomal bypass. The combined deletion of these two enzymes, however, does not result in a fully lethal photorespiratory phenotype, indicating even more redundancy in the photorespiratory HP-into-glycerate conversion. Here, we report on a third enzyme, HPR3 (At1g12550), in Arabidopsis (Arabidopsis thaliana), which also reduces HP to glycerate and shows even more activity with glyoxylate, a more upstream intermediate of the photorespiratory cycle. The deletion of HPR3 by T-DNA insertion mutagenesis results in slightly altered leaf concentrations of the photorespiratory intermediates HP, glycerate, and glycine, indicating a disrupted photorespiratory flux, but not in visible alteration of the phenotype. On the other hand, the combined deletion of HPR1, HPR2, and HPR3 causes increased growth retardation, decreased photochemical efficiency, and reduced oxygen-dependent gas exchange in comparison with the hpr1xhpr2 double mutant. Since in silico analysis and proteomic studies from other groups indicate targeting of HPR3 to the chloroplast, this enzyme could provide a compensatory bypass for the reduction of HP and glyoxylate within this compartment.     

The microbial degradation of cyclohexanecarboxylic acid by a beta-oxidation pathway with simultaneous induction to the utilization of benzoate

The metabolism of cyclohexanecarboxylic acid by a bacterium, designated PRL W19, follows a pathway involving beta-oxidation of coenzyme A intermediates analogous to the classical oxidation of fatty acids. The organism appears to have the property for the constitutive metabolism of caproic acid, and cell extracts contain high levels of the enzymes required for the functioning of the fatty acid cycle. However, the metabolism of cyclohexanecarboxylic acid requires induction by growth or incubation with an appropriate substrate. Extracts of induced cells contain several enzyme activities which are synthesized in response to the induction process. These enzymes include cyclohexanecarboxyl-CoA synthetase, cyclohexanecarboxyl-CoA dehydrogenase, 1-cyclohexenecarboxyl-CoA hydratase, and trans-2-hydroxycyclohexanecarboxyl-CoA dehydrogenase. A characteristics feature of this organism is that it becomes induced for the metabolism of benzoate and catechol during growth on cyclohexanecarboxylic acid, but benzoate does not appear to be an obligatory intermediate in the metabolism of cyclohexanecarboxylic acid.     

Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni.

Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name 'quinohaemoprotein alcohol dehydrogenase'. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.