Properties of an inducible C 4 -dicarboxylic acid transport system in Bacillus subtilis

The transport of the tricarboxylic acid cycle C(4)-dicarboxylic acids was studied in both the wild-type strain and tricarboxylic acid cycle mutants of Bacillus subtilis. Active transport of malate, fumarate, and succinate was found to be inducible by these dicarboxylic acids or by precursors to them, whereas glucose or closely related metabolites catabolite-repressed their uptake. l-Malate was found to be the best dicarboxylic acid transport inducer in succinic dehydrogenase, fumarase, and malic dehydrogenase mutants. Succinate and fumarate are accumulated over 100-fold in succinic dehydrogenase and fumarase mutants, respectively, whereas mutants lacking malate dehydrogenase were unable to accumulate significant quantities of the C(4)-dicarboxylic acids. The stereospecificity of this transport system was studied from a comparison of the rates of competitive inhibition of both succinate uptake and efflux in a succinate dehydrogenase mutant by utilizing thirty dicarboxylic acid analogues. The system was specific for the C(4)-dicarboxylic acids of the tricarboxylic acid cycle, neither citrate nor alpha-ketoglutarate were effective competitive inhibitors. Of a wide variety of metabolic inhibitors tested, inhibiors of oxidative phosphorylation and of the formation of proton gradients were the most potent inhibitors of transport. From the kinetics of dicarboxylic acid transport (K(m) approximately 10(-4) M for succinate or fumarate in succinic acid dehydrogenase and fumarase mutants) and from the competitive inhibition studies, it was concluded that an inducible dicarboxylic acid transport system mediates the entry of malate, fumarate, or succinate into B. subtilis. Mutants devoid of alpha-ketoglutarate dehydrogenase were shown to accumulate both alpha-ketoglutarate and glutamate, and these metabolites subsequently inhibited the transport of all the C(4)-dicarboxylic acids, suggesting a regulatory role.     

Polymyxin B biosynthesis and tricarboxylic acid cycle enzymatic activity]

Activity of pyruvate dehydrogenase (PDG), isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase and malate dehydrogenase was found in the extracts of the cells of Bac. polymyxa 153, an organism producing polymyxin B. Dependence of the activity of the above enzymes on the carbon source in the medium, aeration conditions, strain features and culture age was shown. A low level of polymyxin B biosynthesis was observed at high activity of PDG and dehydrogenases of the tricarbonic acid cycle. Increased antibiotic production was recorded against the background of decreases values of the above enzyme activities.     

Inhibition of mitochondrial alpha-ketoglutarate dehydrogenase by 1-methyl-4-phenylpyridinium ion

Effects of 1-methyl-4-phenylpyridinium ion (MPP+) on the activities of NAD+- or NADP+-linked dehydrogenases in the TCA cycle were studied using mitochondria prepared from mouse brains. Activities of NAD+- and NADP+-linked isocitrate dehydrogenases, NADH- and NADPH-linked glutamate dehydrogenases, and malate dehydrogenase were little affected by 2 mM of MPP+. However, alpha-ketoglutarate dehydrogenase activity was significantly inhibited by MPP+. Kinetic analysis revealed a competitive type of inhibition. Inhibition of alpha-ketoglutarate dehydrogenase may be one of the important mechanisms of MPP+-induced inhibition of mitochondrial respiration, and of neuronal degeneration.     

Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.     

Kinetic advantages of hetero-enzyme complexes with glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex

We have found previously (Fahien, L.A., Kmiotek, E.H., MacDonald, M. J., Fibich, B., and Mandic, M. (1988) J. Biol. Chem. 263, 10687-10697) that glutamate-malate oxidation can be enhanced by cooperative binding of mitochondrial aspartate aminotransferase and malate dehydrogenase to the alpha-ketoglutarate dehydrogenase complex. The present results demonstrate that glutamate dehydrogenase, which forms binary complexes with these enzymes, adds to this ternary complex and thereby increases binding of the other enzymes. Kinetic evidence for direct transfer of alpha-ketoglutarate and NADH, within these complexes, has been obtained by measuring steady-state rates of E2 when most of the substrate or coenzyme is bound to the aminotransferase or glutamate dehydrogenase (E1). Rates significantly greater than those which can be accounted for by the concentration of free ligand, calculated from the measured values of the E1-ligand dissociation constants, require that the E1-ligand complex serve as a substrate for E2 (Srivastava, D. K., and Bernhard, S. A. (1986) Curr. Tops. Cell Regul. 28, 1-68). By this criterion, NADH is transferred directly from glutamate dehydrogenase to malate dehydrogenase and alpha-ketoglutarate is channeled from the aminotransferase to both glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex. Similar evidence indicates that GTP bound to an allosteric site on glutamate dehydrogenase functions as a substrate for succinic thiokinase. The potential physiological advantages to channeling of activators and inhibitors as well as substrates within multienzyme complexes organized around the alpha-ketoglutarate dehydrogenase complex are discussed.     

Trichomonas vaginalis: characterization of its glutamate dehydrogenase

An NADP-linked glutamate dehydrogenase (EC 1.4.1.4) was found in the soluble fraction of Trichomonas vaginalis. Its molecular weight was about 230,000 (gel filtration). The enzyme, partially purified by diafiltration and hydroxyapatite column chromatography, was heat stable (1 hr at 57 C). It catalyzed both the amination of alpha-ketoglutarate (mean Km 0.6 mM) and the deamination of glutamate (mean Km 1.2 mM) The optimum pH of the amination reaction was 6.7, and that of the deamination reaction was 8. Glutamate was a competitive inhibitor of the amination reaction (mean Ki 5.6 mM) and alpha-ketoglutarate a partially competitive inhibitor of the deamination reaction (mean Ki 0.45 mM). Both guanosine and inosine diphosphates (1 mM) increased the Km alpha-ketoglutarate fivefold (mean Ki's 0.3 and 0.4 mM, respectively). Guanosine diphosphate reduced the Km glutamate 40%. Adenosine di- and triphosphate (1 mM) were ineffective. Because the amination reaction displayed substrate inhibition, guanosine and inosine diphosphates were potent natural inhibitors, and ammonia released by deamination reactions would tend to raise pH (amination operative at acid pH), we hypothesize that the deamination reaction may predominate in the living organism.     

Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate.

The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.     

Mutagen Stability of Alkylation-Sensitive Mutants of Bacillus subtilis

The phosphotransacetylase of Veillonella alcalescens catalyzes a reversible reaction with Michaelis-Menten kinetics for all substrates. The rate of the reverse reaction (the synthesis of acetyl coenzyme A from acetyl phosphate) was 6.5 times greater than the rate of the forward reaction (the synthesis of acetyl phosphate from acetyl coenzyme A). The apparent K(m) values determined for the forward reaction were 8.6 x 10(-6)m for acetyl coenzyme A and 9.3 x 10(-3)m for phosphate. In the reverse reaction, the K(m) values were 3.3 x 10(-4)m for coenzyme A and 5.9 x 10(-4)m for acetyl phosphate. The results of an analysis of the inhibition by end products in the forward and reverse directions were compatible with a random bi- bi- mechanism. The enzyme was inhibited by adenosine triphosphate and adenosine diphosphate but was not affected by reduced nicotinamide adenine dinucleotide or pyruvate. The inhibition by adenosine triphosphate was noncompetitive with respect to acetyl phosphate and competitive with respect to coenzyme A. MgCl(2) reversed the inhibition by adenosine triphosphate or adenosine diphosphate. The role of Mg(2+) and adenylates in the regulation of phosphotranscetylase activity is discussed.     

Affinity labeling of the inhibitory DPNH site of bovine liver glutamate dehydrogenase by 5'-fluorosulfonylbenzoyl adenosine.

A new adenosine analogue has been synthesized, 5'-fluorosulfonylbenzoyl adenosine, which reacts covalently with bovine liver glutamate dehydrogenase with the incorporation of approximately 1 mol of 5'-sulfonylbenzoyl adenosine per peptide chain. Native glutamate dehydrogenase is known to be inhibited by relatively high concentrations of DPNH by binding to a second noncatalytic site; the major change in the kinetic characteristics of the modified enzyme is a total loss of this inhibition by DPNH. The modified enzyme retains full catalytic activity as measured in the absence of allosteric ligands, is still inhibited more than 90% by GTP, and is activated normally by ADP. These results demonstrate that the catalytic as well as the GTP and ADP regulatory sites are distinct from the inhibitory DPNH site. The rate constant for reaction of 5'-fluorosulfonylbenzoyl adenosine is decreased by high concentrations of DPNH alone or by DPNH plus GTP, but not by the substrate alpha-ketoglutarate, the coenzymes DPN or TPNH, or the regulators ADP or GTP alone. These observations are consistent with the postulate that the 5'-fluorosulfonylbenzoyl adenosine attacks exclusively the second inhibitory DPNH site. The DPNH inhibition is abolished when an average of only 0.5 mol of 5'-sulfonylbenzoyl adenosine per peptide chain has been incorporated. The structure of 5'-fluorosulfonylbenzoyl adenosine is critical in determining the course of the modification reaction. The smaller compound p-fluorosulfonylbenzoic acid does not affect the kinetic characteristics of the enzyme, and the isomeric compound 3'-fluorosulfonylbenzoyl adenosine produces a different pattern of changes in the regulatory properties (Pal. P. K., Wechter, W. J., and Colman, R. F. (1975) Biochemistry 14, 707-715). Indeed, enzyme which has combined stoichiometrically with 5'-fluorosulfonylbenzoyl adenosine is still able to react with 3'-fluorosulfonylbenzoyl adenosine; thus, the two adenosine analogues appear to react at distinct sites on glutamate dehydrogenase. It is proposed that 5'-fluorosulfonylbenzoyl adenosine will be complementary to 3'-fluorosulfonylbenzoyl adenosine as a general affinity label for dehydrogenases as well as other classes of enzymes which use adenine nucleotides as substrates or regulators.     

Bacteriophages of Bacillus polymyxa

Virulent bacteriophages of colistin--producing Bacillus polymyxa strains were studied. The phages were found to differ in lytic spectrum and were active only against strains of B. polymyxa. They did not attack other strains of the genus Bacillus. The virulent bacteriophages belong to two morphological groups differing in size. The size of the DNA of the bacteriophages of both groups is similar and ranges from 74.9 X 10(6) to 87.8 X 10(6) daltons. The cells of different B. polymyxa strains were also found to carry various defective phages which could be shown after mitomycin C induction of cell cultures. The antibacterial activity of mitomycin C induced cell lysates was not detected. Strains of B. polymyxa most probably devoid of defective bacteriophages (delysogenized) were isolated.     

Correlation of the effects of citric acid cycle metabolites on succinate oxidation by rat liver mitochondria and submitochondrial particles.

1. Succinate dehydrogenase is inhibited by citrate and beta-hydroxy-butyrate in a complex manner, both in mitochondria and submitochondrial particles. Kinetics of inhibition in the particles points to a competitive component in the mechanism involved. 2. Pyruvate, alpha-ketoglutarate, malate, and glutamate stimulate oxidation of succinate by mitochondria. 3. Stimulation by alpha-ketoglutarate and glutamate is not influenced by the presence of rotenone. 4. Stimulation by pyruvate is higher in the absence of rotenone and increases significantly in the presence of K+ and valinomycin. Pyruvate supplies in mitochondria reducing equivalents for malate dehydrogenase operating in the reverse direction-reduction of oxaloacetate to malate. 5. Stimulation by malate is higher in the presence of rotenone.     

Keto acid metabolism in Desulfovibrio.

Four strains of Desulfovibrio each excreted pyruvate to a constant level during growth; it was re-absorbed when the substrate (lactate) was exhausted. Malate, succinate, fumarate and malonate also accumulated during growth. One of the strains (Hildenborough) excreted alpha-ketoglutarate as well as pyruvate when incubated in nitrogen-free medium; the former was re-absorbed on addition of NH4Cl. In a low-lactate nitrogen-free medium, strain Hildenborough rapidly re-absorbed the pyruvate initially excreted, but did not re-absorb the alpha-ketoglutarate. Arsenite (I mM) prevented the accumulation of alpha-ketoglutarate; I mM-malonate did not affect the accumulation of keto acids. Isocitrate dehydrogenase activity (NAD-specific) in all strains was lower than NADP-specific glutamate dehydrogenase activity. Alpha-Ketoglutarate dehydrogenase could not be detected in any strain. NADPH oxidase activity was demonstrated. This and previous work indicate that a tricarboxylic acid pathway from citrate to alpha-ketoglutarate exists in Desulfovibrio spp., and that succinate can be synthesized via malate and fumarate; however, an intact tricarboxylic acid cycle is evidently not present. The findings are compared with observations on biosynthetic pathways in clostridia, obligate lithotrophs, phototrophs, and methylotrophs, and various facultative bacteria.     

Regulation of insulin release by factors that also modify glutamate dehydrogenase

Leucine and monomethyl succinate initiate insulin release, and glutamine potentiates leucine-induced insulin release. Alanine enhances and malate inhibits leucine plus glutamine-induced insulin release. The insulinotropic effect of leucine is at least in part secondary to its ability to activate glutamate oxidation by glutamate dehydrogenase (Sener, A., Malaisse-Lagae, F., and Malaisse, W. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5460-5464). The effect of these other amino acids or Krebs cycle intermediates on insulin release also correlates with their effects on glutamate dehydrogenase and their ability to regulate inhibition of this enzyme by alpha-ketoglutarate. For example, glutamine enhances insulin release and islet glutamate dehydrogenase activity only in the presence of leucine. This could be because leucine, especially in the presence of alpha-ketoglutarate, increases the Km of glutamate and converts alpha-ketoglutarate from a noncompetitive to a competitive inhibitor of glutamate. Thus, in the presence of leucine, this enzyme is more responsive to high levels of glutamate and less responsive to inhibition by alpha-ketoglutarate. Malate could decrease and alanine could increase insulin release because malate increases the generation of alpha-ketoglutarate in islet mitochondria via the combined malate dehydrogenase-aspartate aminotransferase reaction, and alanine could decrease the level of alpha-ketoglutarate via the alanine transaminase reaction. Monomethyl succinate alone is as stimulatory of insulin release as leucine alone, and glutamine enhances the action of both. Succinyl coenzyme A, leucine, and GTP are all bound in the same region on glutamate dehydrogenase, where GTP is a potent inhibitor and succinyl coenzyme A and leucine are comparable activators. Thus, the insulinotropic properties of monomethyl succinate could result from it increasing the level of succinyl coenzyme A and decreasing the level of GTP via the succinate thiokinase reaction.     

Zn2+ inhibits alpha-ketoglutarate-stimulated mitochondrial respiration and the isolated alpha-ketoglutarate dehydrogenase complex

Intracellular free Zn(2+) is elevated in a variety of pathological conditions, including ischemia-reperfusion injury and Alzheimer's disease. Impairment of mitochondrial respiration is also associated with these pathological conditions. To test whether elevated Zn(2+) and impaired respiration might be linked, respiration of isolated rat liver mitochondria was measured after addition of Zn(2+). Zn(2+) inhibition (K(i)(app) = approximately 1 micrometer) was observed for respiration stimulated by alpha-ketoglutarate at concentrations well within the range of intracellular Zn(2+) reported for cultured hepatocytes. The bc(1) complex is inhibited by Zn(2+) (Link, T. A., and von Jagow, G. (1995) J. Biol. Chem. 270, 25001-25006). However, respiration stimulated by succinate (K(i)(app) = approximately 6 micrometer) was less sensitive to Zn(2+), indicating the existence of a mitochondrial target for Zn(2+) upstream from bc(1) complex. Purified pig heart alpha-ketoglutarate dehydrogenase complex was strongly inhibited by Zn(2+) (K(i)(app) = 0.37 +/- 0.05 micrometer). Glutamate dehydrogenase was more resistant (K(i)(app) = 6 micrometer), malate dehydrogenase was unaffected, and succinate dehydrogenase was stimulated by Zn(2+). Zn(2+) inhibition of alpha-ketoglutarate dehydrogenase complex required enzyme cycling and was reversed by EDTA. Reversibility was inversely related to the duration of exposure and the concentration of Zn(2+). Physiological free Zn(2+) may modulate hepatic mitochondrial respiration by reversible inhibition of the alpha-ketoglutarate dehydrogenase complex. In contrast, extreme or chronic elevation of intracellular Zn(2+) could contribute to persistent reductions in mitochondrial respiration that have been observed in Zn(2+)-rich diseased tissues.     

Properties of malate dehydrogenase isolated from Methanospirillum hungatii.

A NADH-linked oxygen-tolerant malate dehydrogenase was purified 270-fold from cell extracts of Methanospirillum hungatii. Inhibitors of the enzyme included ADP, alpha-ketoglutarate, and excess NADH. Inhibition patterns for ADP were competitive with respect to NADH and non-competitive with respect to oxalacetate. Inhibition by alpha-ketoglutarate was non-competitive with oxalacetate as variable substrate and uncompetitive with respect to NADH. alpha-Ketoglutarate is surmised to function as an end-product inhibitor of the enzyme in reactions converting oxalacetate to alpha-ketoglutarate. No enzyme activity was detected in the direction of malate conversion to oxalacetate, in keeping with a strictly biosynthetic function of the enzyme. An analysis of variance of intial rate data fit to sequential and ping-pong equations showed that a sequential mechanism was perferred. The malate dehydrogenase of M. hungatii resembles those of many other bacteria and eucaryotic cells respect to molecular weight (61,700) and reaction mechanism, but may be regulated differently.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

Interaction between NAD-dependent isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex, and NADH:ubiquinone oxidoreductase

Interaction between the alpha-ketoglutarate dehydrogenase complex and NAD+-dependent isocitrate dehydrogenase was detected with a variety of techniques including polyethylene glycol precipitation, ultracentrifugation, and centrifugal gel filtration on a Sepharose 6B column. The interaction was specific in that citrate synthase, cytosolic malate dehydrogenase, and NADP-dependent isocitrate dehydrogenase did not interact with alpha-ketoglutarate dehydrogenase complex. The interaction was not inhibited by either 0.1 M KCl or 0.4 M (NH4)2SO4, but was completely prevented by 5% glycerol. A new method for the preparation of NADH: ubiquinone oxidoreductase resulted in an enzyme having a protein subunit composition similar to that of classical complex I preparation. Evidence is given for the existence of ternary complexes containing NADH:ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-NAD-dependent isocitrate dehydrogenase and NADH: ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-succinate thiokinase. These data suggest that a part of the citric acid cycle may be located in the vicinity of NADH: ubiquinone oxidoreductase. These complexes may facilitate the transport of metabolites among these enzymes without their equilibrating with the whole compartment.     

Components required for the formation of CH-4 from methylcobalamin by extracts of Methanobacillus omelianskii

Wood, J. M. (University of Illinois, Urbana), and R. S. Wolfe. Components required for the formation of CH(4) from methylcobalamin by extracts of Methanobacillus omelianskii. J. Bacteriol. 92:696-700. 1966.-Optimal conditions for the formation of CH(4) from methylcobalamin (methyl-cobalt-5,6-dimethylbenzimidazolylcobamide) by partially purified extracts of Methanobacillus omelianskii (Methanobacterium omelianskii) were achieved by the addition of two protein fractions, adenosine triphosphate, Mg(++), and a reduced flavin adenine dinucleotide-generating system. Adenosine diphosphate and adenosine monophosphate play an important role in the regulation of C(1) transfer in this reaction. The possible position of adenosine triphosphate in B(12)-dependent methyl transfer is discussed.     

Catabolism of alpha-ketoglutarate by a sucA mutant of Bradyrhizobium japonicum: evidence for an alternative tricarboxylic acid cycle

A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acid substrates malate, succinate, and fumarate. However, a Bradyrhizobium japonicum sucA mutant that is missing alpha-ketoglutarate dehydrogenase is able to grow on malate as its sole source of carbon. This mutant also fixes nitrogen in symbiosis with soybean, where dicarboxylic acids are its principal carbon substrate. Using a flow chamber system to make direct measurements of oxygen consumption and ammonium excretion, we confirmed that bacteroids formed by the sucA mutant displayed wild-type rates of respiration and nitrogen fixation. Despite the absence of alpha-ketoglutarate dehydrogenase activity, whole cells of the mutant were able to decarboxylate alpha-[U-(14)C]ketoglutarate and [U-(14)C]glutamate at rates similar to those of wild-type B. japonicum, indicating that there was an alternative route for alpha-ketoglutarate catabolism. Because cell extracts from B. japonicum decarboxylated [U-(14)C]glutamate very slowly, the gamma-aminobutyrate shunt is unlikely to be the pathway responsible for alpha-ketoglutarate catabolism in the mutant. In contrast, cell extracts from both the wild type and mutant showed a coenzyme A (CoA)-independent alpha-ketoglutarate decarboxylation activity. This activity was independent of pyridine nucleotides and was stimulated by thiamine PP(i). Thin-layer chromatography showed that the product of alpha-ketoglutarate decarboxylation was succinic semialdehyde. The CoA-independent alpha-ketoglutarate decarboxylase, along with succinate semialdehyde dehydrogenase, may form an alternative pathway for alpha-ketoglutarate catabolism, and this pathway may enhance TCA cycle function during symbiotic nitrogen fixation.