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PubMed1990

Enzyme Activities Affecting End Product Distribution by Lactobacillus plantarum in Response to Changes in pH and O(2).

Lactobacillus plantarum catabolic end products changed in response to environmental conditions. While lactate was always the major end product, acetate was produced in alkaline and aerobic environments. Acetoin levels decreased under alkaline conditions. Changes in acetoin dehydrogenase, acetate kinase, NADH oxidase, pyruvate oxidase, and acetate kinase activities correlated with changes in end product distribution.

Journal Article↗
PubMed1989

Oxygen activation and defence against oxygen toxicity in a psychrophilic Bacteroidaceae.

When suddenly exposed to air the growth of the obligate anaerobic bacterium of the bacteroidaceae type, strain B6, continues for a few hours before coming to a complete stop. When air is shut off soon after growth has ceased, the organism is able to reestablish anaerobic conditions due to an ability to reduce O2, and resumes normal growth after another few hours. The O2 reducing ability of the organism is due to the presence in the cells of a particle-bound NADH oxidase, a soluble NADPH oxidase and a soluble pyruvate oxidase. The two pyridine nucleotide oxidases reduce O2 to H2O2, the pyruvate oxidase reduces O2 to H2O. Catalase and peroxidase were not detected in anaerobically grown cells. Kinetic studies with cell-free extracts showed that the pyruvate oxidase had a considerably greater affinity (smaller Km) for O2 and capacity (higher Vmax) for O2 reduction than the two other oxidases. It is postulated that the pyruvate oxidase acts as a scavenger for O2, leading to the non-toxic reduction product H2O, and thus functions as a defense mechanism against oxygen toxicity when the organism is exposed to aerobic condition.

Air Microbiology↗
PubMed2002

Enzymatic assay of gamma-cystathionase activity using pyruvate oxidase-peroxidase sequential reaction.

A highly sensitive method has been developed for the determination of gamma-cystathionase (EC. 4.4.1.1.) activity in rat tissues using beta-chloro-L-alanine as a substrate. This method is based on colorimetry for the determination of pyruvate produced from beta-chloro-L-alanine with the beta-elimination catalyzed by gamma-cystathionase, coupling a color enzymatic reaction with pyruvate oxidase and peroxidase. The absorbance increases with the oxidized color of a leuco dye, N-(carboxymethylamino)-4,4'-bis (dimethylamino)-diphenylamine at 727 nm is proportional to the gamma-cystathionase activity. The present method is more sensitive and more rapid than the usual methods and does not require troublesome steps such as centrifugation. The calibration curve is linear up to 1.6 microg of partially purified enzyme (100 U/l). Comparison with the usual method with L-homoserine as a substrate gave good correlation (r=0.990). The present method was applied to the determination of gamma-cystathionase activity in adult male rat tissues. The mean activities in liver and kidney were 8.03 and 3.91 U/g wet weight (n=10), respectively.

Animals↗
PubMed2005

Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses.

In a series of previous reports it was established by implementing metabolic flux, NMR/MS, and Northern blot analysis that the glyoxylate shunt, the TCA cycle, and acetate uptake by acetyl-CoA synthetase are more active in Escherichia coli BL21 than in Escherichia coli JM109. These differences were accepted as the reason for the differences in the glucose metabolism and acetate excretion of these two strains. Examination of the bacterial metabolism by microarrays and time course Northern blot showed that in addition to the glyoxylate shunt, the TCA cycle and the acetate uptake, other metabolic pathways are active differently in the two strains. These are gluconeogenesis, sfcA shunt, ppc shunt, glycogen biosynthesis, and fatty acid degradation. It was found that in E. coli JM109, acetate is produced by pyruvate oxidase (poxB) using pyruvate as a substrate rather than by phosphotransacetylase-acetate kinase (Pta-AckA) system which uses acetyl-CoA. The inactivation of the gluconeogenesis enzyme phosphoenolpyruvate synthetase (ppsA), the activation of the anaplerotic sfcA shunt, and low and stable pyruvate dehydrogenase (aceE, aceF) cause pyruvate accumulation which is converted to acetate by pyruvate oxidase B. The behavior of the ppsA, acs, and aceBAK in JM109 was dependent on the glucose supply strategy. When the glucose concentration was high, no transcription of these genes was observed and acetate concentration increased, but at low glucose concentrations these genes were expressed and the acetate concentration decreased. It is possible that there is a major regulatory molecule that controls not only ppsA and aceBAK but also acs. The gluconeogenesis pathway (fbp, pckA, and ppsA) which leads to glycogen accumulation is constitutively active in E. coli BL21 regardless of glucose feeding strategy.

Acetates↗
PubMed2003

Serine metabolism in Lactobacillus plantarum.

This study investigated the metabolism of (L-) serine by Lactobacillus plantarum B3089 isolated from cheese. Serine was deaminated by growing cells to ammonia with the corresponding formation of acetate and formate. Serine was also deaminated by non-growing cells to ammonia but with the formation of acetate only (no production of formate). Phosphoserine and threonine were not catabolised. It is proposed that serine was deaminated by serine dehydratase (deaminase) to ammonia and pyruvate. Pyruvate was further catabolised predominantly to acetate, carbon dioxide and formate in growing cells, catalysed by pyruvate-formate lyase and pyruvate oxidase; some of the pyruvate was converted to acetoin. In non-growing cells, however, pyruvate-formate lyase was inactive and pyruvate oxidase degraded the pyruvate to acetate and carbon dioxide. Serine dehydratase activity could not be detected in cell-free extracts, presumably because of enzyme instability. The growth of L. plantarum was neither enhanced nor stimulated by serine under the current conditions. Whereas there was little difference in serine utilisation between pH 7.0 and pH 5.8, serine utilisation was decreased by 30% at pH 5.0. NaCl of up to 4% (w/v) concentration had little effect on serine utilisation. Serine had no impact on lactose metabolism. Lactose was fermented mainly to lactate (73%) with the remainder converted to an unidentified polysaccharide (27%).

Cheese↗
PubMed1987

Pleiotropic effects of poxA regulatory mutations of Escherichia coli and Salmonella typhimurium, mutations conferring sulfometuron methyl and alpha-ketobutyrate hypersensitivity.

A transposon Tn10 insertion into the Salmonella typhimurium poxA gene was identified among a set of mutations conferring sulfometuron methyl (SM) hypersensitivity. This Tn10 insertion mapped to 95 min on the S. typhimurium chromosome, a location analogous to that of poxA in the Escherichia coli genome. Like the E. coli poxA mutant, this mutant had reduced pyruvate oxidase activity, reduced cross-reacting material to antiserum to purified E. coli pyruvate oxidase, and reduced growth rates. In addition, the following phenotypes were identified for the E. coli and S. typhimurium poxA mutants: hypersensitivity to SM and alpha-ketobutyrate (AKB), deficiency in AKB metabolism, reduced activity of acetolactate synthase, and hypersensitivity to a wide range of bacterial growth inhibitors, including antibiotics, amino acid analogs, and dyes. An E. coli mutant defective in poxB, the structural gene encoding pyruvate oxidase, did not have these phenotypes; therefore, they are not solely a consequence of a pyruvate oxidase deficiency. Comparisons were made with mutant alleles of two other genes that are located near poxA and confer related phenotypes. The S. typhimurium poxA mutant differed both genetically and phenotypically from an miaA mutant. E. coli abs mutants had somewhat reduced pyruvate oxidase activity but had normal AKB metabolism. The relationship of the pleiotropic phenotypes of the poxA mutants to their SM hypersensitivity is discussed.

Acetolactate Synthase↗
PubMed1991

Biosensor system for continuous flow determination of enzyme activities. II. Simultaneous determination of plural enzyme activities.

A biosensor system for continuous flow determination of plural enzyme activities was prepared from the combination of two pyruvate sensors, a prereactor and a flow cell. This system was applied to the simultaneous determination of lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities in the same sample. These enzyme activities can be determined by measuring pyruvate produced by the enzyme reactions as follows. The amount of pyruvic acid can also be determined from the amount of oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. (Formula: see text). Therefore, both of the detectors for the determination of lactic dehydrogenase and glutamic-pyruvic transaminase activities were prepared from the combination of a pyruvate oxidase membrane and an oxygen electrode. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate. A linear relation was obtained between the output current and LDH or GPT activities in the range of 50 to 3,600 IU l-1 or 6 to 1,000 IU l-1, respectively. Each assay of these enzyme activities was completed within 15 min. The results obtained had a precision of ca. 4%. The sensor was stable for more than 25 days at 5 degrees C.

Alanine Transaminase↗
PubMed1996

Isolation and characterization of subunits of acetohydroxy acid synthase isozyme III and reconstitution of the holoenzyme.

The separately cloned large and small subunits of AHAS isozyme III from Escherichia coli have been isolated and purified. The essentially pure small subunit (17 kDa ilvH product) was obtained by a procedure exploiting its low solubility. The large, catalytic subunit (62 kDa ilvI product) was isolated by standard techniques, to > or = 95% purity. The large subunit has low catalytic activity relative to holoenzyme (approximately 5%) but shows similar substrate specificity and qualitatively similar cofactor dependence and inhibition by a sulfonylurea herbicide. Its activity is insensitive to valine, and the protein does not bind valine. The small subunit binds valine with Kd = 0.2 mM. Reconstitution of the holoenzyme from its subunits leads to a complex with the properties of the native protein, including valine inhibition of activity with Ki = 12 microM. Reconstitution titrations confirm the 1:1 stoichiometry of subunit assembly and a tendency to dissociation (about 50% dissociation near 0.1 microM subunit). Size exclusion HPLC indicates that either subunit alone is largely monomeric, and that assembly of the holoenzyme (two large + two small subunits, 150-160 kDa) requires FAD. On the basis of its homology with pyruvate oxidase and pyruvate decarboxylase, we suggest that the active sites of AHAS III are located at the interface of a dimer of catalytic subunits. Our experiments suggest that such a dimer is not stable except in the presence of the small subunits. The association of valine with sites on the regulatory subunits presumably influences the active sites by an allosteric conformational effect.

Acetolactate Synthase↗
PubMed1993

Growth of rho 0 human Namalwa cells lacking oxidative phosphorylation can be sustained by redox compounds potassium ferricyanide or coenzyme Q10 putatively acting through the plasma membrane oxidase.

Pyruvate is conventionally used as a key growth supplement for mammalian rho 0 cells that lack mitochondrial DNA and are thereby devoid of oxidative phosphorylation. We have tested the proposition that cultured rho 0 human cells can be grown using redox compounds other than pyruvate. The results show that potassium ferricyanide and coenzyme Q10 can each be used to replace pyruvate to support the growth of rho 0 Namalwa cells (a lymphoblastoid cell line). Ferricyanide and coenzyme Q10 have both been reported as substrates for a plasma membrane NADH oxidase system which is capable of re-oxidising cytosolic NADH to NAD+. These compounds are also known to stimulate the activity of this enzyme system. We interpret our data to indicate that redox support for growth of rho 0 human cells can be achieved by external electron acceptors such as ferricyanide (a plasma membrane impermeant compound), or coenzyme Q10 (an integral component of the plasma membrane oxidase), through the enhanced conversion of cytosolic NADH to NAD+. This re-oxidation of NADH enables glycolysis to function efficiently as the sole source of cellular ATP, in the absence of mitochondrial oxidative phosphorylation in rho 0 cells. This has important implications for the development of new strategies for the amelioration of the bioenergy decline that occurs in mitochondrial disease and during the human ageing process.

Cell Division↗
PubMed2006

Engineering Escherichia coli to improve culture performance and reduce formation of by-products during recombinant protein production under transient intermittent anaerobic conditions.

Three E. coli strains, named VAL22, VAL23, and VAL24, were engineered at the level of mixed-acid fermentation pathways to improve culture performance under transient anaerobic conditions. VAL22 is a single mutant with an inactivated poxB gene that codes for pyruvate oxidase which converts pyruvate to acetate. VAL23 is a double mutant unable to produce lactate and formate due to deletions of the ldhA and pflB genes that code for lactate dehydrogenase and pyruvate-formate lyase, respectively. VAL24 is a triple mutant with ldhA and pflB deleted and poxB inactivated. Engineered strains were cultured under oscillating dissolved oxygen tension (DOT) in a scale-down system, to simulate gradients occurring in large-scale bioreactors. Kinetic and stoichiometric parameters of constant (10%) and oscillating DOT cultures of the engineered strains were compared with those of the parental strain, W3110. All strains expressed recombinant green fluorescent protein (GFP) as a protein model. Mutant strains showed improved specific growth rate, reduced by-product formation, and reduced specific glucose uptake rate compared to the parental strain, when cultured under oscillating DOT. In particular, lactate and formate production was abolished and acetate accumulation was reduced by 9-12%s. VAL24 showed the best performance, as specific growth and GFP production rates, and maximum GFP concentration were not affected by DOT gradients and were at least twofold higher than those of W3110 under constant DOT. Under oscillating DOT, VAL24 wasted about 40% less carbon into fermentation by-products than W3110. It was demonstrated that, although E. coli responds rapidly to DOT fluctuations by deviating to fermentative metabolism, such pathways can be eliminated as they are not necessary for bacterial survival during the short circulation times typical of large-scale cultures. The approach shown here opens new possibilities for designing metabolically engineered strains, with reduced sensitivity to DOT gradients and improved performance under typical conditions of large-scale cultures.

Acetyltransferases↗
PubMed1989

2-Acetylthiamin pyrophosphate: an enzyme-bound intermediate in thiamin pyrophosphate-dependent reactions.

Several enzymes catalyze reactions that may involve acetylthiamin pyrophosphate (acetyl-TPP) as an intermediate. These enzymes are phosphoketolase, pyruvate oxidase and several pyruvate oxidoreductases. Acetyl-TPP can be synthesized and used as a carrier to analyze quenched reaction mixtures for the presence of [14C]acetyl-TPP. Synthetic acetyl-TPP exhibits unusual chemical properties and a unique pH-rate profile that serves as a powerful means of characterizing [14C]acetyl-TPP that has been isolated from quenched enzymatic reaction mixtures. Using this and other methods, extensive evidence has been obtained for the involvement of acetyl-TPP in certain reactions catalyzed by the pyruvate dehydrogenase complex (PDH complex) of Escherichia coli. Acetyl-TPP is chemically competent as an intermediate in the decarboxylation and dehydrogenation of pyruvate by the PDH complex; and it is transiently formed during the course of this reaction. It may be an enzyme-bound intermediate or it may be in equilibrium with such an intermediate. Acetyl-TPP is very likely to be an intermediate of the phosphoketolase reaction. However, no direct evidence linking it to the phosphoketolase reaction mechanism is yet available. It is unclear whether acetyl-TPP is an intermediate in the pyruvate oxidoreductase reactions. In one example, that of the ketoacid oxidoreductase of Halobacterium halobium, analysis by electron paramagnetic resonance spectroscopy indicates the involvement of a hydroxyethyl-TPP-radical as an intermediate. It is unknown whether the subsequent reaction of this radical with coenzyme A an an oxidized FeS cluster to produce acetyl coenzyme A and the reduced cluster involves the intermediate formation of acetyl-TPP.

Aldehyde-Lyases↗
PubMed2000

Crystal versus solution structures of thiamine diphosphate-dependent enzymes.

The quaternary structures of the thiamine diphosphate-dependent enzymes transketolase (EC 2.2.1.1; from Saccharomyces cerevisiae), pyruvate oxidase (EC 1.2.3.3; from Lactobacillus plantarum), and pyruvate decarboxylase (EC 4.1.1.1; from Zymomonas mobilis and brewers' yeast, the latter in the native and pyruvamide-activated forms) were examined by synchrotron x-ray solution scattering. The experimental scattering data were compared with the curves calculated from the crystallographic models of these multisubunit enzymes. For all enzymes noted above, except the very compact pyruvate decarboxylase from Z. mobilis, there were significant differences between the experimental and calculated profiles. The changes in relative positions of the subunits in solution were determined by rigid body refinement. For pyruvate oxidase and transketolase, which have tight intersubunit contacts in the crystal, relatively small modifications of the quaternary structure (root mean square displacements of 0.23 and 0.27 nm, respectively) sufficed to fit the experimental data. For the enzymes with looser contacts (the native and activated forms of yeast pyruvate decarboxylase), large modifications of the crystallographic models (root mean square displacements of 0.58 and 1.53 nm, respectively) were required. A clear correlation was observed between the magnitude of the distortions induced by the crystal environment and the interfacial area between subunits.

Artifacts↗
PubMed2003

Nanoporous aluminum oxide as a novel support material for enzyme biosensors.

To construct novel amperometric sensors for the detection of hydrogen peroxide and pyruvate, peroxidase and pyruvate oxidase were immobilized in self-supporting nanoporous alumina membranes those made by anodic oxidation. Pyruvate oxidase and other enzymes were enclosed in poly(carbamoylsulfonate) hydrogel and sucked into the nanoporous alumina structure before polymerization. The alumina membranes were investigated by scanning electron microscopy before and after the enzyme immobilization. In an amperometric flow detector cell, pyruvate and hydrogen peroxide were detected under flow injection analysis conditions in concentration ranges from 1 microM to 100 microM and 5 microM to 500 microM, respectively. The achieved operational stability showed that alumina membranes can be used to construct enzyme-modified electrodes.

Aluminum Oxide↗
PubMed2003

The phosphonopyruvate decarboxylase from Bacteroides fragilis.

The Bacteroides fragilis capsular polysaccharide complex is the major virulence factor for abscess formation in human hosts. Polysaccharide B of this complex contains a 2-aminoethylphosphonate functional group. This functional group is synthesized in three steps, one of which is catalyzed by phosphonopyruvate decarboxylase. In this paper, we report the cloning and overexpression of the B. fragilis phosphonopyruvate decarboxylase gene (aepY), purification of the phosphonopyruvate decarboxylase recombinant protein, and the extensive characterization of the reaction that it catalyzes. The homotrimeric (41,184-Da subunit) phosphonopyruvate decarboxylase catalyzes (kcat = 10.2 +/- 0.3 s-1) the decarboxylation of phosphonopyruvate (Km = 3.2 +/- 0.2 microm) to phosphonoacetaldehyde (Ki = 15 +/- 2 microm) and carbon dioxide at an optimal pH range of 7.0-7.5. Thiamine pyrophosphate (Km = 13 +/- 2 microm) and certain divalent metal ions (Mg(II) Km = 82 +/- 8 microm; Mn(II) Km = 13 +/- 1 microm; Ca(II) Km = 78 +/- 6 microm) serve as cofactors. Phosphonopyruvate decarboxylase is a member of the alpha-ketodecarboxylase family that includes sulfopyruvate decarboxylase, acetohydroxy acid synthase/acetolactate synthase, benzoylformate decarboxylase, glyoxylate carboligase, indole pyruvate decarboxylase, pyruvate decarboxylase, the acetyl phosphate-producing pyruvate oxidase, and the acetate-producing pyruvate oxidase. The Mg(II) binding residue Asp-260, which is located within the thiamine pyrophosphate binding motif of the alpha-ketodecarboxylase family, was shown by site-directed mutagenesis to play an important role in catalysis. Pyruvate (kcat = 0.05 s-1, Km = 25 mm) and sulfopyruvate (kcat approximately 0.05 s-1; Ki = 200 +/- 20 microm) are slow substrates for the phosphonopyruvate decarboxylase, indicating that this enzyme is promiscuous.

Amino Acid Sequence↗
PubMed2005

Development of a disposable pyruvate biosensor to determine pungency in onions (Allium cepa L.).

A disposable prototype pyruvate biosensor was constructed using pyruvate oxidase immobilised on mediated meldolas blue electrodes to determine pungency in onions (Allium cepa L.). The optimum operating potential was +150 mV (versus Ag/AgCl). A strong correlation between the biosensor response and untreated onion juice of known pyruvate concentration 2-12 micromol/g fresh weight (FW) was demonstrated. The biosensor was able to differentiate between low and high pungency onions. The detection limit using 1 unit of pyruvate oxidase was 1-2 micromol/g FW. Optimum concentrations of co-factors TPP, FAD and MgSO4 comprising the enzyme cocktail were determined as being 0.04, 0.1 and 30 mM, respectively.

Biosensing Techniques↗
PubMed2006

The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography.

Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase.

Binding Sites↗
PubMed2007

The lysine decarboxylase CadA protects Escherichia coli starved of phosphate against fermentation acids.

Conflicting results have been reported for the rate and extent of cell death during a prolonged stationary phase. It is shown here that the viability of wild-type cells (MG1655) could decrease >or=10(8)-fold between days 1 and 14 and between days 1 and 6 of incubation under aerobic and anaerobic phosphate (P(i)) starvation conditions, respectively, whereas the cell viability decreased moderately under ammonium and glucose starvation conditions. Several lines of evidence indicated that the loss of viability of P(i)-starved cells resulted primarily from the catabolism of glucose into organic acids through pyruvate oxidase (PoxB) and pyruvate-formate lyase (PflB) under aerobic and anaerobic conditions, respectively. Weak organic acids that are excreted into the medium can reenter the cell and dissociate into protons and anions, thereby triggering cell death. However, P(i)-starved cells were efficiently protected by the activity of the inducible GadABC glutamate-dependent acid resistance system. Glutamate decarboxylation consumes one proton, which contributes to the internal pH homeostasis, and removes one intracellular negative charge, which might compensate for the accumulated weak acid anions. Unexpectedly, the tolerance of P(i)-starved cells to fermentation acids was markedly increased as a result of the activity of the inducible CadBA lysine-dependent acid resistance system that consumes one proton and produces the diamine cadaverine. CadA plays a key role in the defense of Salmonella at pH 3 but was thought to be ineffective in Escherichia coli since the protection of E. coli challenged at pH 2.5 by lysine is much weaker than the protection by glutamate. CadA activity was favored in P(i)-starved cells probably because weak organic acids slowly reenter cells fermenting glucose. Since the environmental conditions that trigger the death of P(i)-starved cells are strikingly similar to the conditions that are thought to prevail in the human colon (i.e., a combination of low levels of P(i) and oxygen and high levels of carbohydrates, inducing the microbiota to excrete high levels of organic acids), it is tempting to speculate that E. coli can survive in the gut because of the activity of the GadABC and CadBA glutamate- and lysine-dependent acid resistance systems.

Acetates↗