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PubMed1969

Phosphorylation and the reduced nicotinamide adenine dinucleotide oxidase reaction in Streptococcus agalactiae.

Cell-free extracts from aerobically grown Streptococcus agalactiae cells possess a reduced nicotinamide adenine dinucleotide (NADH) oxidase which is linked to oxygen. It is inhibited by cyanide, although cytochromes evidently are not involved. Adenosine triphosphate (ATP) formation occurs during the reaction, but 66 to 75% of the total ATP is formed nonoxidatively. The remaining 25 to 35% of the ATP formation is related to the oxidation of NADH. The formation of ATP in the oxidative reaction can be prevented by excluding oxygen or adding cyanide to prevent NADH oxidation. It can also be prevented by adding methylene blue or pyruvate, which bypasses electron transport to oxygen, but does not interfere with NADH oxidation. Potential sources of ATP, such as glycolysis, the pyruvate oxidase reaction, or the oxidative pentose cycle, are not present, and the high nonoxidative ATP formation is ascribed to the adenylate kinase reaction. The reaction requires adenosine diphosphate (ADP) as a phosphate acceptor. NADH oxidation is independent of ADP. Antimycin A, amytal, and 2,4-dinitrophenol decreased, but did not prevent, oxidative formation of ATP. P:O ratios ranged from 0.15 to 0.25. All of the oxidative activity was in the soluble portion of the cell-free extracts.

Adenosine Triphosphatases↗
PubMed1980

Oxygen utilization by Lactobacillus plantarum. II. Superoxide and superoxide dismutation.

Cell-free extracts of Lactobacillus plantarum contain non-proteinaceous compounds which mimic superoxide dismutase activity. Using the test system in which O-2 is generated by xanthine oxidase, superoxide dismutase activity is found in cell-free extracts, where proteins are removed by precipitation. This activity is strongly decreased after dialysis of cell-free extracts. Superoxide dismutase activity was also investigated by means of pulse radiolysis. Cell-free extracts of Escherichia coli were also investigated as a comparison, which were known to contain superoxide dismutase. With cell-free extracts of both L. plantarum and E. coli the decay of O-2 was markedly increased. However, the type of reaction of the O-2 decay was of first order in the presence of E. coli extracts due to superoxide dismutase(s), and of second order in the presence of L. plantarum extracts, indicating that O-2 elimination is not an enzymic reaction. Mn2+ phosphate(s) might be responsible for the observed elimination of O-2. The production of O-2 is not detectable during NADH-, lactate- or pyruvate oxidase reactions in L. plantarum extracts.

Cell-Free System↗
PubMed2004

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme.

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

Acetolactate Synthase↗
PubMed1996

Homology modeling of the structure of bacterial acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis.

Acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and condensation of the resulting two-carbon moiety with a second alpha-keto acid. It belongs to a family of homologous, TPP-dependent enzymes which catalyze different reactions which start from decarboxylation of alpha-keto acids. A model for the structure of Escherichia coli AHAS isozyme II, based on its homology with pyruvate oxidase and experimental testing of the model by site-directed mutagenesis, has been used here to study how AHAS controls the chemical fate of a decarboxylated keto acid. Because of the potential conformational freedom of the reacting substrates, residues interacting with the substrate could not be identified directly from the model of AHAS. Three residues were considered as candidates for involvement in the recognition of alpha-ketobutyrate, as the amino acids at these sites in a unique low-specificity AHAS are different from those in typical AHASs, which are highly specific for reaction with alpha-ketobutyrate as second substrate, in preference to pyruvate. These residues were altered in AHAS II by site-directed mutagenesis. Replacement of Trp464 lowers the specificity by at least 1 order of magnitude, with minor effects on the activity or stability of the enzyme, suggesting that Trp464 contributes > or = 1.3 kcal mol-1 to interaction with the "extra" methyl of alpha-ketobutyrate. Mutations of Met460 or Thr70 have small effects on specificity and do affect other properties of the protein. A model for enzyme-substrate interactions can be proposed on the basis of these results. The model of AHAS also explains previously reported spontaneous mutants of AHAS resistant to sulfonylurea herbicides, which probably bind in the narrow depression which provides access to the bound TPP. A role for the C terminus of the enzyme polypeptide in determination on the reaction pathway is also possible.

Acetolactate Synthase↗
PubMed2006

Pyruvate:quinone oxidoreductase in Corynebacterium glutamicum: molecular analysis of the pqo gene, significance of the enzyme, and phylogenetic aspects.

Corynebacterium glutamicum recently has been shown to possess pyruvate:quinone oxidoreductase (PQO), catalyzing the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the electron acceptor. Here, we analyze the expression of the C. glutamicum pqo gene, investigate the relevance of the PQO enzyme for growth and amino acid production, and perform phylogenetic studies. Expression analyses revealed that transcription of pqo is initiated 45 bp upstream of the translational start site and that it is organized in an operon together with genes encoding a putative metal-activated pyridoxal enzyme and a putative activator protein. Inactivation of the chromosomal pqo gene led to the absence of PQO activity; however, growth and amino acid production were not affected under either condition tested. Introduction of plasmid-bound pqo into a pyruvate dehydrogenase complex-negative C. glutamicum strain partially relieved the growth phenotype of this mutant, indicating that high PQO activity can compensate for the function of the pyruvate dehydrogenase complex. To investigate the distribution of PQO enzymes in prokaryotes and to clarify the relationship between PQO, pyruvate oxidase (POX), and acetohydroxy acid synthase enzymes, we compiled and analyzed the phylogeny of respective proteins deposited in public databases. The analyses revealed a wide distribution of PQOs among prokaryotes, corroborated the hypothesis of a common ancestry of the three enzymes, and led us to propose that the POX enzymes of Lactobacillales were derived from a PQO.

Amino Acids↗
PubMed1986

A carnitine/acylcarnitine translocase assay applicable to biopsied muscle specimens without requiring mitochondrial isolation.

A simple method for assaying the mitochondrial carnitine/acylcarnitine translocase of muscles that needs only few milligrams of fresh tissue is described. The procedure involves monitoring of the sulphobetaine (an inhibitor of the translocase)-sensitive acetylation of sub-saturating concentrations of carnitine in the medium, linked to the oxidation of [2-14C]pyruvate in the presence of malonate. Conditions affecting the reliability of the outlined procedure and the ancillary information to be collected, namely the activities of pyruvate oxidase system and carnitine acetyltransferase, for detecting possible deficiency of the translocase are described, together with data on the translocase activity in human skeletal muscle, in rat red and white skeletal muscles and in rat heart. The concepts outlined should allow development of assays of other mitochondrial transporters that also would require neither isolation of mitochondria nor availability of a large quantity of tissue, both of which are otherwise needed at present.

Acetylation↗
PubMed1989

Enzymic urea assay: a new colorimetric method based on hydrogen peroxide measurement.

We describe a new enzymic colorimetric method in which urea is measured in serum by use of a single reagent mixture. Ammonia produced by urea hydrolysis, catalyzed by urease, reacts with glutamate and ATP in the presence of glutamine synthetase. The ADP so produced is assayed in reactions catalyzed sequentially by pyruvate kinase and pyruvate oxidase in a system that generates hydrogen peroxide. The hydrogen peroxide is measured at 500 or 550 nm in a reaction catalyzed by horseradish peroxidase, with phenol/4-aminophenazone as the chromogen. The reaction is complete in 15 min at 37 degrees C. The standard curve is linear up to a urea concentration of 40 mmol/L. Precision is good; CVs ranged from 2.5% to 3.1%. Results by the present method compared well with those by a candidate Reference Method and are not subject to interferences from commonly used drugs and anticoagulants.

Adenosine Diphosphate↗
PubMed2005

Effect of lpdA gene knockout on the metabolism in Escherichia coli based on enzyme activities, intracellular metabolite concentrations and metabolic flux analysis by 13C-labeling experiments.

The lipoamide dehydrogenase (LPD) encoded by lpdA gene is a component of the pyruvate dehydrogenase complex (PDHc), alpha-ketoglutarate dehydrogenase (AKGDH) and the glycine cleavage multi-enzyme (GCV) systems. In the present study, cell growth characteristics, enzyme activities and intracellular metabolite concentrations were compared between the parent strain Escherichia coli BW25113 and its lpdA knockout mutant in batch and continuous cultures. The lpdA knockout mutant produced significantly more pyruvate and L-glutamate under aerobiosis. Some D-lactate and succinate also accumulated in the culture broth. Based on the investigation of enzyme activities and intracellular metabolite concentrations, acetyl-CoA was considered to be formed by the combined reactions through pyruvate oxidase (PoxB), acetyl-CoA synthetase (Acs) and acetate kinase (Ack)-phosphoacetyltransferase (Pta) in the lpdA mutant. The effect of the lpdA gene knockout on the intracellular metabolic flux distributions was investigated based on 1H-13C NMR spectra and GC-MS signals obtained from 13C-labeling experiment using the mixture of [U-13C] glucose, [1-13C] glucose, and naturally labeled glucose. Flux analysis of the lpdA mutant indicated that the Entner-Doudoroff (ED) pathway and the glyoxylate shunt were activated. The fluxes through glycolysis and oxidative pentose phosphate (PP) pathway (except for the flux through glucose-6-phosphate dehydrogenase) were slightly downregulated. The TCA cycle was also downregulated in the mutant strain. On the other hand, the fluxes through the anaplerotic reactions of PEP carboxylase, PEP carboxykinase and malic enzyme were upregulated, which were consistent with the results of enzyme activities. Furthermore, the influence of the poxB gene knockout on the growth of E. coli was also studied because of its similar function to PDHc which connects the glycolysis to the TCA cycle. Under aerobiosis, a comparison of lpdA mutant and poxB mutant indicated that PDHc is the main enzyme which catalyzes the reaction from pyruvate to acetyl-CoA in the parent strain, while PoxB plays a very important role in the PDHc-deficient strain.

Biological Transport↗
PubMed1975

Active transport in Excherichia coli B membrane vesicles. Irreversible uncoupling by chloropyruvate.

In the accompanying report (Kaczorowski, G., Shaw, L., Laura, R., and Walsh, C. (1975) J. Biol. Chem. 250, 8921-8930), we have shown that the oxidation of beta-chloro-D-alanine by a membrane-bound D-alanine dehydrogenase results in the inactivation of both dehydrogenase-coupled and P-enolpyruvate-dependent active transport in membrane vesicles. We have also demonstrated that chemically prepared chloropyruvate has the same inactivating effects on transport. In this report, we show that in addition to abolishing hexose and proline uptake, chloropyruvate inhibits lactose and several other amino acid uptake systems to different extents, although proline transport is the most severely inhibited. The degree of transport inactivation also depends on whether the keto acid is added exogenously or is generated by the D-alanine dehydrogenase. Chloropyruvate treatment does not inhibit D-alanine dehydrogenase, D-lactate dehydrogenase of the passage of electrons to oxygen by the membrane cytochrome chain. However, alanine racemase and pyruvate oxidase (to a lesser extent) are inactivated by this keto acid. Treatment of vesicles with chloropyruvate does not affect the establishment of maintenance of a membrane potential, however, this does inhibit solute transport in response to an artificially induced potential. If chloropyruvate is added at any point during a time course of proline transport, there is an instantaneous blockade of amino acid uptake suggesting that the proline carrier can no longer translocate solute across the membrane. Upon examining the functionality of the carrier protein after exposure to chloropyruvate, there is no appreciable difference in efflux or exchange properties as compared to untreated controls. Therefore chloropyruvate does not block proline passage through the membrane, but rather appears to interfere with the ability of the proline carrier to sense the membrane potential. The beta-halo keto acid does not then uncouple respiration from energization of the membrane but does interfere with the ability of the energized membrane state to be used for the transport of most solutes.

Alanine↗
PubMed2003

Towards a phosphoproteome map of Corynebacterium glutamicum.

In this study, the phosphoproteome of Corynebacterium glutamicum, an industrially important soil bacterium of the Corynebacterium/Mycobacterium/Nocardia (CMN) group of Gram-positive bacteria, was investigated by two different detection methods: first, by in vivo radio-labeling using [(33)P]-phosphoric acid with subsequent autoradiography and second, by immunostaining with phosphoamino acid-specific monoclonal antibodies. After two-dimensional gel electrophoresis (2-DE), around 60 [(33)P]-labeled protein spots were visualized and around 90 antibody-decorated protein spots detected; 31 of the protein spots were detected with both methods. By peptide mass fingerprinting, 41 different proteins were identified, namely 5-enolpyruvylshikimate 3-phosphate synthase, aconitase, acyl-CoA carboxylase, acyl-CoA synthetase, ATP (synthase alpha- and beta-chain), carbamoyl-phosphate synthase, citrate synthase, cysteine synthase, DnaK, the elongation factors G, P, Ts and Tu, enolase, fructose bisphosphate aldolase, fumarase, Gap dehydrogenase, glutamine synthetase I, glycine hydroxymethyltransferase, GroEL2, GTPase, heat-inducible transcriptional repressor DnaJ2, inorganic pyrophosphatase, isocitrate dehydrogenase, ketol-acid reductoisomerase, lactate dehydrogenase, leucine-tRNA ligase, lipoamide dehydrogenase, methionine synthase, O-acetylhomoserine sulfhydrylase, pyruvate carboxylase, pyruvate kinase, pyruvate oxidase, ribosomal protein S1, RNA polymerase (beta-subunit), succinyl-CoA:CoA transferase, transketolase and UDP-N-acetylmuramoyl-L-alanine ligase, besides a hypothetical 35k protein and a hypothetical glucose kinase. Both detection techniques were used to create a phosphoproteome map. Additionally, the influence of nitrogen deprivation on the phosphoproteome of C. glutamicum was investigated.

Corynebacterium↗
PubMed1996

Rational molecular design and genetic engineering of herbicide resistant crops by structure modeling and site-directed mutagenesis of acetohydroxyacid synthase.

Plants with specific resistance to a single class of herbicides have been genetically engineered by introduction of genes encoding rationally designed mutant acetohydroxyacid synthase (AHAS) enzymes. Suitable substitution mutations were identified from a three-dimensional model of an AHAS-inhibitor complex. The structural model was generated based on homology to pyruvate oxidase and an imidazolinone inhibitor was positioned in the proposed binding site using structure-activity data for this class of herbicide. Biochemical analysis of the mutant proteins expressed in Escherichia coli enabled iterative optimization of the mutant genes. Expression of recombinant proteins in tobacco plants conferred resistance in vivo. The novel approach coupling molecular modeling and molecular biology has many advantages over traditional random mutagenesis and selection methods and will be crucial to the future development for environmentally safe and sustainable agricultural systems.

Acetolactate Synthase↗
PubMed2000

Characterization of abundance and diversity of lactic acid bacteria in the hindgut of wood- and soil-feeding termites by molecular and culture-dependent techniques.

Lactic acid bacteria have been identified as typical and numerically significant members of the gut microbiota of Reticulitermes flavipes and other wood-feeding lower termites. We found that also in the guts of the higher termites Nasutitermes arborum (wood-feeding), Thoracotermes macrothorax, and Anoplotermes pacificus (both soil-feeding), lactic acid bacteria represent the largest group of culturable carbohydrate-utilizing bacteria (3.6-5.2x10(4) bacteria per gut; 43%-54% of all colonies). All isolates were coccoid and phenotypically difficult to distinguish, but their enterobacterial repetitive intergenic consensus sequence (ERIC) fingerprint patterns showed a significant genetic diversity. Six different genotypes each were identified among the isolates from R. flavipes and T. macrothorax, and representative strains were selected for further characterization. By 16S rRNA gene sequence analysis, strain RfL6 from R. flavipes was classified as a close relative of Enterococcus faecalis, whereas strain RfLs4 from R. flavipes and strain TmLO5 from T. macrothorax were closely related to Lactococcus lactis. All strains consumed oxygen during growth on glucose and cellobiose; oxygen consumption of these and other isolates from both termite species was due to NADH and pyruvate oxidase activities, but did not result in H2O2 formation. In order to assess the significance of the isolates in the hindgut, denaturing gradient gel electrophoresis was used to compare the fingerprints of 16S rRNA genes in the bacterial community of R. flavipes with those of representative isolates. The major DNA band from the hindgut bacterial community was further separated by bisbenzimide-polyethylene glycol electrophoresis, and the two resulting bands were sequenced. Whereas one sequence belonged to a spirochete, the second sequence was closely related to the sequences of the Lactococcus strains RfLs4 and TmLO5. Apparently, those isolates represent strains of a new Lactococcus species which forms a significant fraction of the complex hindgut community of the lower termite R. flavipes and possibly also of other termites.

Animals↗
PubMed1987

A rapid assay method for ammonia using glutamine synthetase from glutamate-producing bacteria.

A rapid enzymatic assay method for ammonia was developed by using glutamine synthetase from glutamate-producing bacteria together with pyruvate kinase, lactate dehydrogenase, and NADH. The time required for determination of 25 nmol of ammonia was 5 min with 1 unit of glutamine synthetase, as opposed to 14-30 min with 1 unit of glutamate dehydrogenases from various sources. The present method was used to determine ammonia in serum, microbiol-culture broth, and waste water. The method can be modified for spectrophotometry in the visible region by substituting pyruvate oxidase, peroxidase, and appropriate chromogens for lactate dehydrogenase and NADH. With 4-aminoantipyrine (4AA) and phenol, and with 4AA and N-ethyl-N-2-hydroxyethyl-m-toluidine as chromogens, the sensitivity of ammonia determination was 0.65 and 1.7 times that with glutamate dehydrogenase, respectively. The present method was also applicable to the continuous detection of the activity of some ammonia-forming enzymes such as guanase, adenosine deaminase, and urease and to the determination of 0.5-30 microM ATP-ADP after some modification of the mixture.

Adenosine Diphosphate↗
PubMed1986

A rapid method for isolation of synaptosomes on Percoll gradients.

A new rapid method for fractionation of crude synaptosomes (postmitochondrial pellet, P2) on a discontinuous 4-step Percoll gradient is described. The homogeneity and integrity of the 5 major subcellular fractions were determined by analysis of the distribution of protein, lactate dehydrogenase, cytochrome oxidase, pyruvate dehydrogenase, synapsin I (a synaptic vesicle marker) and the myelin basic proteins. The biochemical results were substantiated by quantitative electron microscopy. Fractions 3, 4 and 5 were enriched in synaptosomes and contained 19.7, 40.6 and 19.5% of the intact, identifiable synaptosomes in P2, respectively. Fraction 1 was enriched in membranous material, fraction 2 in myelin and fraction 5 in extrasynaptosomal mitochondria. The synaptosomes in fractions 3, 4 and 5 differed in their size, and their content of mitochondria, synapsin I and neurotransmitters. These results suggest that partial separation of different pools of synaptosomes has been achieved. The synaptosomes in fractions 3, 4 and 5 are viable, as they take up calcium, phosphate and noradrenaline; they are metabolically normal as judged by their ability to perform protein phosphorylation and they respond normally to depolarization by increasing calcium uptake, protein phosphorylation and neurotransmitter release. The synaptosomes in fraction 4 are relatively homogeneous and appear to be free of contamination from lysed synaptosomes and synaptic plasma membranes. This constitutes a major advantage of the Percoll method over traditional procedures which involve centrifugation to equilibrium. We have therefore confirmed (J. Neurochem., 43 (1984) 1114-1123) the advantages of Percoll use over traditional procedures, while further reducing the time taken, and extended our analysis to show that the present procedure provides a fractionation of synaptosomes into different pools of viable synaptosomes.

Animals↗