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Results for “Pyruvate Oxidase”

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PubMed2001

Competence regulation by oxygen availability and by Nox is not related to specific adjustment of central metabolism in Streptococcus pneumoniae.

In Streptococcus pneumoniae oxygen availability is a major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in cultures grown aerobically. The implication of oxidative metabolism and more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in nox, which encodes NADH oxidase, did not change this trait. Consistently, mRNA levels of glyceraldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable to wild-type levels, as did the specific activities of key enzymes which control central metabolism. Competence regulation by oxygen involving the NADH oxidase activity is not due to significant modification of carbon flux through glycolysis. Failure to obtain loss-of-function mutation in L-ldh, which encodes the L-lactate dehydrogenase, indicates its essential role in pneumococci whatever their growth status.

Blotting, Northern↗

PubMed1989

Pyruvate decarboxylase is like acetolactate synthase (ILV2) and not like the pyruvate dehydrogenase E1 subunit.

Protein sequences of pyruvate decarboxylase (PDC) derived from cloned yeast (Saccharomyces cerevisiae) and bacterial (Zymomonas mobilis) genes were compared with each other and with sequence databases. Extensive sequence similarities were found between them and with two others: cytochrome-linked pyruvate oxidase from Escherichia coli and acetolactate synthase (ilvI in E. coli; ILV2 gene in S. cerevisiae). All catalyse decarboxylation of pyruvate using thiamine pyrophosphate (TPP) as cofactor. General overall similarity suggests common ancestry for these enzymes. None of the sequences was similar to the E1 component of pyruvate dehydrogenase from E. coli which also decarboxylates pyruvate with the help of TPP.

Acetolactate Synthase↗

PubMed2006

Fed-batch two-phase production of alanine by a metabolically engineered Escherichia coli.

DL-Alanine was produced from glucose in an Escherichia coli pfl pps poxB ldhA aceEF pTrc99A-alaD strain which lacked pyruvate-formate lyase, phosphoenolpyruvate (PEP) synthase, pyruvate oxidase, lactate dehydogenase, components of the pyruvate dehydogenase complex and over-produced alanine dehydrogenase (ALD). A two-phase process was developed with cell growth under aerobic conditions followed by alanine production under anaerobic conditions. Using the batch mode, cells grew to 5.3 g/l in 9 h with the accumulation of 6-10 g acetate/l, and under subsequent anaerobic conditions achieved 34 g alanine/l in 13 h with a yield of 0.86 g/g glucose. Using the fed-batch mode at micro = 0.15 h(-1), only about 1 g acetate/l formed in the 25 h required for the cells to reach 5.6 g/l, and 88 g alanine/l accumulated during the subsequent 23 h. This fed-batch process attained an alanine volumetric productivity of 4 g/lh during the production phase, and a yield that was essentially 1 g/g.

Aerobiosis↗

PubMed2001

Amperometric determination of acetic acid with a trienzyme/poly(dimethylsiloxane)-bilayer-based sensor.

A trienzyme sensor for the amperometric determination of acetic acid was prepared by immobilizing acetate kinase (AK), pyruvate kinase (PK), and pyruvate oxidase (PyOx) on a poly(dimethylsiloxane) (PDMS)-coated electrode. AK catalyzes the phospho-transferring reaction between acetic acid and ATP to form ADP; PK, the phospho-transferring reaction between ADP and phosphoenolpyruvate to form pyruvic acid; and PyOx, the oxidation of pyruvic acid with oxygen. The oxygen consumption could be monitored by using the PDMS-coated electrode without interference from the PyOx reaction product, hydrogen peroxide. Thus, the concentration of acetic acid (5 microM-0.5 mM) could be determined from the decrease in the cathodic current at -0.4 V vs Ag/AgCl. This is the first example of a biosensor that can be used for the determination of acetic acid in ethanol-containing food samples. The acetate-sensing electrode could be used for more than one month.

Acetate Kinase↗

PubMed1998

Activation of thiamin diphosphate in enzymes.

Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

Allosteric Regulation↗

PubMed2006

[Characterization of hydrogen peroxide production by a novel oral streptococci, S. oligofermentans isolated from human oral cavity].

High level of hydrogen peroxide was produced by S . oligofermentans, a novel oral streptococcus isolated from human oral cavity previously in our laboratory. To characterize the hydrogen peroxide production by S. oligofermentans, hydrogen peroxide yields at different growth phases and from different substrates were assayed. The results turned out that hydrogen peroxide production started at the beginning of logarithmic phase, and reached the maximal yield at the early stationary phase. Peptone and yeast extract could be the main substrates for hydrogen peroxide production. Moreover, lactate, as a fermentative product of glucose, can be another substrate for hydrogen peroxide production. Furthermore three oxidases activities possibly associated with H2O2 production were assayed, and both activities of lactate oxidase and NADH oxidase were detected under aerobic condition, while pyruvate oxidase activity was not detected in the permeabilized cells of S. oligofermentans, implying that S. oligofermentans could mainly rely on the two enzymes activities for H2O2 production.

Humans↗

PubMed1991

Oxygen and the sugar metabolism in oral streptococci.

Streptococci have several ways of adapting themselves to the constantly changing environment of the human oral cavity. This paper discusses the adaptation of sugar metabolism to variations in oxygen levels. In all streptococci the Embden-Meyerhof pathway of glycolysis works under aerobic as well as anaerobic conditions, but pyruvate is converted into different metabolic end products depending on the oxygen levels. Under anaerobic conditions all streptococci form formate, acetate, and ethanol by a pyruvate formate-lyase pathway. If sugar is in excess, they also form lactate using a lactate dehydrogenase. Under aerobic conditions pyruvate formate-lyase is inactivated. This enzyme is then replaced by a pyruvate oxidase in some streptococci and by a pyruvate dehydrogenase in others. The characteristics of these enzymes help streptococci like S. sanguis, S. oralis, S. gordonii, and S. mitis to compete successfully with other bacteria in those sites of the oral cavity that are freely exposed to saliva, while mutans streptococci have to colonize anaerobic sites such as those in-between the teeth and in the occlusal fissures of the teeth.

Glucose↗

PubMed1994

Reconstitution of functional electron transfer between membrane biological elements in a two-dimensional lipidic structure at the electrode interface.

These studies develop a methodology to form supported phospholipid bilayers at an electrode/solution interface that models biological membrane systems. Two kinds of electrode were used, a planar gold electrode and a microporous aluminium oxide electrode on which octadecanethiol or octadecyltrichlorosilane was self-assembled. The supported lipidic structures were produced by transfer of a phospholipid monolayer by the Langmuir-Blodgett technique or by direct fusion of phospholipid vesicles. Ubiquinone was introduced into the lipidic structures during their formation; electrochemical measurements demonstrated the mobility of ubiquinone along the plane of the bilayer. A membrane enzyme, pyruvate oxidase from E. coli, was successfully incorporated into this artificial bilayer and was found to be able to exchange electrons with ubiquinone present in the bilayer.

Electrochemistry↗

PubMed1996

Lactate and acetate production in Listeria innocua.

Listeria innocua NCTC 11289 was grown aerobically in continuous culture in defined media at 30 degrees C. Both acetate and lactate were produced, the proportion of acetate decreased with increasing dilution rate. Enzymatic analysis showed lactate dehydrogenase was activated 10-fold by fructose-1, 6-bisphosphate. The presence of phosphate acetyltransferase and acetate kinase but not pyruvate oxidase was detected, suggesting the sequential action of phosphate acetyltransferase and acetate kinase to produce acetate from acetyl CoA via acetylphosphate.

Acetate Kinase↗

PubMed2006

Interference between Streptococcus pneumoniae and Staphylococcus aureus: In vitro hydrogen peroxide-mediated killing by Streptococcus pneumoniae.

The bactericidal activity of Streptococcus pneumoniae toward Staphylococcus aureus is mediated by hydrogen peroxide. Catalase eliminated this activity. Pneumococci grown anaerobically or genetically lacking pyruvate oxidase (SpxB) were not bactericidal, nor were nonpneumococcal streptococci. These results provide a possible mechanistic explanation for the interspecies interference observed in epidemiologic studies.

Antibiosis↗

PubMed2004

Transcription levels of key metabolic genes are the cause for different glucose utilization pathways in E. coli B (BL21) and E. coli K (JM109).

Acetate accumulation is a common problem observed in aerobic high cell density cultures of Escherichia coli. It has been hypothesized in previous reports that the glyoxylate shunt is active in E. coli BL21, the low acetate producer, and inactive in E. coli JM109, the high acetate producer. This hypothesis was further strengthened by incorporating 13C from uniformly labeled glucose into TCA cycle intermediates. Using northern blot analyses, the current report demonstrates that the reason for the inactivity of the glyoxylate pathway in E. coli JM109 is the no apparent transcription of isocitrate lyase (aceA) and malate synthase (aceB), and transcription of the isocitrate lyase repressor (iclR). The reverse is seen in E. coli BL21 where the glyoxylate pathway is active due to constitutive transcription of aceA and aceB and no transcription of the iclR. In addition, there is a difference between the two strains in the transcription of the acetyl-CoA synthetase (acs), phosphotransacetylase-acetate kinase (pta-ackA) pathway, and pyruvate oxidase (poxB), pathway. The transcript of acs is higher in E. coli BL21 and lower in the E. coli JM109, while the reverse is true for poxB transcription.

Acetate Kinase↗

PubMed1987

Two enzymatic methods for determination of the phosphate content of phosphoproteins.

Two new enzymatic methods are described for the determination of the amount of inorganic phosphate covalently bound to macromolecules. In the first method, the inorganic matter obtained after ashing is incubated with [1-14C] pyruvate in the presence of pyruvate oxidase and the enzymatically formed 14CO2 is collected. The other method uses glycogenolytic enzymes to generate NADH which is then measured fluorometrically. The use of these two phosphate-dependent enzymes allows the measurement of phosphate at up to 1000 times greater sensitivity than is obtainable with the phosphomolybdic acid method, thus permitting phosphate analyses to be performed on microgram quantities of proteins.

Molybdenum↗

PubMed2006

Examination of Lactobacillus plantarum lactate metabolism side effects in relation to the modulation of aeration parameters.

AIMS: The characterization of global aerobic metabolism of Lactobacillus plantarum LP652 under different aeration levels, in order to optimize acetate production kinetics and to suppress H2O2 toxicity. METHODS AND RESULTS: Cultures of L. plantarum were grown on different aeration conditions. After sugar exhaustion and in the presence of oxygen, lactate was converted to acetate, H2O2 and carbon dioxide with concomitant ATP production. Physiological assays were performed at selected intervals in order to assess enzyme activity and vitality of the strain during lactic acid conversion. The maximal aerated condition led to fast lactate-to-acetate conversion kinetics between 8 and 12 h, but H2O2 immediately accumulated, thus affecting cell metabolism. Pyruvate oxidase activity was highly enhanced by oxygen tension and was responsible for H2O2 production after 12 h of culture, whereas lactate oxidase and NADH-dependent lactate dehydrogenase activities were not correlated to metabolite production. Limited NADH oxidase (NOX) and NADH peroxidase (NPR) activities were probably responsible for toxic H2O2 levels in over-aerated cultures. CONCLUSION: Modulating initial airflow led to the maximal specific activity of NOX and NPR observed after 24 h of culture, thus promoting H2O2 destruction and strain vitality at the end of the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimal aeration conditions were determined to minimize H2O2 concentration level during growth on lactate.

Acetates↗

PubMedDate not supplied

Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis.

Two strategies for ATP regeneration during cell-free protein synthesis were applied to the large-scale production and single-column purification of active chloramphenicol acetyl transferase (CAT). Fed-batch reactions were performed on a 5-10 mL scale, approximately 2 orders of magnitude greater than the typical reaction volume. The pyruvate oxidase system produced 104 nmol of active CAT in a 5 mL reaction over the course of 5 h. The PANOx system produced 261 +/- 42 nmol, about 7 mg, of active CAT in a 10 mL reaction over the course of 4 h. The reaction product was purified to apparent homogeneity with approximately 70% yield by a simple affinity chromatography adsorption and elution. To our knowledge, this is the largest amount of actively expressed protein to be reported in a simple, fed-batch cell-free protein synthesis reaction.

Adenosine Triphosphate↗

PubMed2000

Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract.

An inverse correlation between colonization of the human nasopharynx by Streptococcus pneumoniae and Haemophilus influenzae, both common upper respiratory pathogens, has been reported. Studies were undertaken to determine if either of these organisms produces substances which inhibit growth of the other. Culture supernatants from S. pneumoniae inhibited growth of H. influenzae, whereas culture supernatants from H. influenzae had no effect on the growth of S. pneumoniae. Moreover, coculture of S. pneumoniae and H. influenzae led to a rapid decrease in viable counts of H. influenzae. The addition of purified catalase prevented killing of H. influenzae in coculture experiments, suggesting that hydrogen peroxide may be responsible for this bactericidal activity. H. influenzae was killed by concentrations of hydrogen peroxide similar to that produced by S. pneumoniae. Hydrogen peroxide is produced by the pneumococcus through the action of pyruvate oxidase (SpxB) under conditions of aerobic growth. Both an spxB mutant and a naturally occurring variant of S. pneumoniae, which is downregulated in SpxB expression, were unable to kill H. influenzae. A catalase-reversible inhibitory effect of S. pneumoniae on the growth of the respiratory tract pathogens Moraxella catarrhalis and Neisseria meningitidis was also observed. Elevated hydrogen peroxide production, therefore, may be a means by which S. pneumoniae is able to inhibit a variety of competing organisms in the aerobic environment of the upper respiratory tract.

Coculture Techniques↗

PubMed2004

Novel biosensors for quantitative phytic acid and phytase measurement.

Phytase (EC 3.1.3.26) and phytic acid (myo-inositol hexaphosphate) play an important environmental role in poultry industry and have a health aspect in food industry. Novel biosensors have been developed for simple, one step quantitative phytic acid and phytase detection. A system based on the sequentially acting enzyme phytase and pyruvate oxidase (POD) was employed for the development of phytase and phytic acid biosensors. Poly(carbamoylsulphonate) (PCS) hydrogel immobilized POD electrode was applied for the detection of phytase. It was based on the indication of phosphate ions produced by the hydrolysis of phytic acid. The phytase biosensor showed a linear response ranging from 0.5 to 6.0 units/ml. A bi-enzyme sensor based on co-immobilization of phytase and POD was developed for the detection of phytic acid on the basis of amperometric detection of the enzymatically-generated hydrogen peroxide at 0.6 V versus Ag/AgCl. It showed a linear response ranging from 0.2 to 2.0 mM with a detection limit of 0.002 mM.

6-Phytase↗

PubMed2003

Biosensor for rapid phosphate monitoring in a sequencing batch reactor (SBR) system.

A thick-film phosphate biosensor based on hydrogel immobilized pyruvate oxidase (POD) has been developed for rapid phosphate process control monitoring in an experimental sequencing batch reactor (SBR) system. We have employed a phosphate biosensor in an off-line monitoring of phosphate concentrations in a bench scale SBR. Measurements with biosensor show a good correlation (r2=0.98) with those of commercial colorimetric phosphate testing kits. The signal response time was 1 min with a detection limit of 5 microM. The biosensor method showed a good operational stability, needed less experimental procedures and a small sample size (approximately 20 microl). This allows its practical application for rapid phosphate measurements to obtain real time process data in a SBR system.

Bioreactors↗

PubMed2000

Differential protein expression in phenotypic variants of Streptococcus pneumoniae.

Streptococcus pneumoniae undergoes spontaneous phase variation resulting in opaque and transparent colony forms. Differences in colony opacity correlate with differences in virulence: the transparent variants are more capable of colonizing the nasopharynx, whereas the opaque variants show increased virulence during systemic infections. To gain insight into the pathogenesis of pneumococcal disease at the molecular level, protein expression patterns of the phenotypic variants of two pneumococcal strains were compared by high-resolution two-dimensional protein electrophoresis. In comparison with transparent variants, the opaque variants reduced the expression of two proteins and overexpressed one protein. The proteins were identified by mass spectrometric analysis. The protein overexpressed in the opaque phenotype revealed significant homology to elongation factor Ts of Helicobacter pylori. One of the two proteins that were underexpressed in the opaque variants revealed significant homology to the proteinase maturation protein PrtM of Lactocobacillus paracasei, a member of the family of peptidyl-prolyl cis/trans isomerases. A consensus lipoprotein signal sequence suggests that the putative proteinase maturation protein A, designated PpmA, is located at the surface of the pneumococcus and may play a role in the maturation of surface or secreted proteins. The second underexpressed protein was identified as pyruvate oxidase, SpxB. The lower SpxB expression in opaque variants most probably explains the reduced production of hydrogen peroxide, a reaction product of SpxB, in this variant. Since a spxB-defective pneumococcal mutant has decreased ability to colonize the nasopharynx (B. Spellerberg, D. R. Cundell, J. Sandros, B. J. Pearce, I. Idanpaan-Heikkila, C. Rosenow, and H. R. Masure, 1996. Mol. Microbiol. 19:803-813, 1996), our data suggest that SpxB plays an important role in enhancing the ability of transparent variants to efficiently colonize the nasopharynx.

Amino Acid Sequence↗