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Results for “Pyruvate Decarboxylase”

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At least 217 records · Page 12Linked to original sources
PubMed1993

[Spectroscopic study of the structure and intramolecular mobility of yeast pyruvate decarboxylase].

Steady-state and time-resolved fluorimetry were used to study the properties of holo- and apopyruvate decarboxylase (EC 4.1.1.1, PDC) from Brewer's yeast after interaction with substrate (pyruvate), cofactor (thiamine diphosphate, ThDP) and Mg2+ ions. The analysis of the enzyme's intrinsic fluorescence as well as of its complex with the probe 2-(p-toluidinylnaphthalene)-6-sulphonate (TNS) revealed that ThDP was found at the polar region of the PDC active sites, inducing a decrease in the mobility of the protein's nearest surroundings. The fluorescent probe had three different sites of binding to the protein apoform, two of which being located at the catalytic site and having different rotation freedom. The study of the PDC complex with thiochrome pyrophosphate, a ThDP structural analogue, pointed to the occurrence of a non-polar region of the enzyme active site for pyruvate absorption besides the polar region. The binding of pyruvate to the protein does not depend upon the cofactor's binding. On the basis of the fluorescent studies a model of the ThDP and pyruvate arrangement at the PDC active site is suggested.

Anilino Naphthalenesulfonates↗
PubMed1978

Chromium (III) thiamin diphosphate: a new probe of enzyme coenzyme--metal ion binding. Interaction with wheat germ pyruvate decarboxylase.

Chromium (III) thiamin diphosphate (CrTDP) is a substitutionally inert complex which is a physical and kinetic probe of the binding mode of metal ion and coenzyme in thiamin diphosphate (TDP) dependent enzymes. CrTDP is prepared by reaction of aquated Cr (III) and TDP and purifed by ion-exchange chromatography. CrTDP binds to the apoenzyme of wheat germ pyruvate decarboxylase, giving an inactive holoenzyme. Chromium (III) ion binds to the apoenzyme in a manner that suggests, in light of the results with CrTDP, that there are two metal ion binding sites. Extension of the use of CrTDP to other enzymes can give specific information about binding and can introduce an active site reporter group

Apoenzymes↗
PubMed2005

Production of glucoamylase in pyruvate decarboxylase deletion mutants of the yeast Kluyveromyces lactis.

Yeasts are widely used as hosts for the production of diverse heterologous proteins ranging from laboratory scale to industrial scale. The aim of this work is to provide new tools for the production of heterologous proteins in the yeast Kluyveromyces lactis. The promoter of the single gene (KlPDC1) encoding pyruvate decarboxylase is strong, inducible, and responsive to the presence of fermentable sugars and anoxic conditions in this yeast. Expression of KlPDC1 is repressed by ethanol and by autoregulation, a mechanism that involves protein KlPdc1. We constructed a heterologous gene expression cassette for a secreted protein (glucoamylase, GAM) under the control of the KlPDC1 promoter on a stable multicopy plasmid. GAM production by wild-type transformed strains was compared with that of klpdc1-deleted transformants. We obtained higher GAM production in the latter strains, which was due to continued expression of the GAM gene during the stationary phase rather than due to GAM transcription levels higher than the wild-type strains during growth phase. This finding opens new perspectives on the physiology of the stationary phase in K. lactis and suggests the possibility of using high-cell-density approaches for the efficient production of heterologous proteins with this yeast.

Bioreactors↗
PubMed1998

Subunit structure, function and organisation of pyruvate decarboxylases from various organisms.

The nature of the environment of macromolecules influences and determines the state of their overall structure and the extent of binding of specific (cofactors, substrates) or unspecific ligands. How these interactions between enzyme molecules and ligands influence their quaternary structures and, in this way, the realisation of high catalytic activity will be discussed here for the enzyme pyruvate decarboxylase from various organisms: brewer's yeast, brewer's yeast strain, recombinant wild type and site-specific mutants of Saccharomyces cerevisiae, the recombinant wild type of the bacterium Zymomonas mobilis and germinating seeds of the plant Pisum sativum from a structural point of view including both high resolution models from crystal structure analysis and low resolution models from small angle X-ray solution scattering with synchrotron radiation.

Macromolecular Substances↗
PubMed2003

Engineering lactic acid bacteria with pyruvate decarboxylase and alcohol dehydrogenase genes for ethanol production from Zymomonas mobilis.

Lactic acid bacteria are candidates for engineered production of ethanol from biomass because they are food-grade microorganisms that can, in many cases, metabolize a variety of sugars and grow under harsh conditions. In an effort to divert fermentation from production of lactic acid to ethanol, plasmids were constructed to express pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), encoded by the pdc and adhB genes of Zymomonas mobilis, in lactic acid bacteria. Several strains were transformed with the plasmids, and transcription of pdc and adhB was confirmed by northern hybridization analysis of transformants. PDC and ADH enzyme activities were at least 5- to 10-fold lower in these bacteria compared to Escherichia coli transformed with the same plasmid. Glucose fermentations were carried out, and some, but not all, of the transformed strains produced more ethanol than the untransformed parent strains. However, lactic acid was the primary fermentation product formed by all of the transformants, indicating that ADH and PDC activities were insufficient to divert significant carbon flow towards ethanol.

Alcohol Dehydrogenase↗
PubMed1986

Conjugated alpha-keto acids as mechanism-based inactivators of brewer's yeast pyruvate decarboxylase: electronic effects of substituents and detection of a long-lived intermediate.

A series of phenyl substituted E-4-phenyl-2-keto-3-butenoic acid derivatives were synthesized (p-Cl, m-Cl, p-NO2, m-NO2, o-NO2, 3,4-Cl2, 2,6-Cl2, p-CH3O, p-(CH3)2N) and tested as potential irreversible inhibitors of brewer's yeast pyruvate decarboxylase (EC 4.1.1.1). All those derivatives with electron withdrawing substituents were found to be time-dependent inactivators of the enzyme, unlike the p-CH3O- and p-(CH3)2N derivatives. Detailed kinetic studies with the m-nitro derivative (the most potent inhibitor) indicated that this compound formed reversible complexes with the enzyme at two sites (supposed regulatory and catalytic with Ki values of 0.026 and 0.13 mM, respectively) prior to irreversible inactivation of the enzyme. In addition, concurrently with the inactivation, addition of the m-NO2 derivative to the enzyme produced a new VIS absorbance with lambda max near 430 nm. This absorbance was attributed to the enzyme-bound enamine intermediate. The time course of formation and disappearance of the intermediate could be determined and provided detailed information about the mechanism of the enzyme.

Carboxy-Lyases↗
PubMed1999

Evolution of a functionally related lactate dehydrogenase and pyruvate decarboxylase pseudogene complex in maize.

A large proportion of the maize genome is repetitive DNA (60-80%) with retrotransposons contributing significantly to the repetitive DNA component. The majority of retrotransposon DNA is located in intergenic regions and is organized in a nested fashion. Analysis of an 8.2-kb segment of maize genomic DNA demonstrated the presence of three retrotransposons of different reiteration classes in addition to lactate dehydrogenase and pyruvate decarboxylase pseudogenes. Both of the pseudogenes were located within a defective retrotransposon element (LP-like element) which possessed identical long terminal repeats (LTRs) with inverted repeats at each end, a primer binding site, a polypurine tract, and generated a 5-bp target site duplication. A model describing the events leading to the formation of the LP-like element is proposed.

Base Sequence↗
PubMed1985

Pyruvate Decarboxylase from Zea mays L. : 2. Examination of Hysteretic Kinetics.

A significant lag phase was observed in the accumulation of product for the reaction catalyzed by pyruvate decarboxylase (PDC) purified from mature maize kernels. The effects of pH, pyruvate, potassium chloride, PDC concentration, and Mg(2+)-thiamine pyrophosphate upon this lag and upon the observed cooperativity were investigated. PDC preincubated with Mg(2+)-thiamine pyrophosphate for six days had Michaelis-Menten kinetics, a Hill number of 1, and no apparent lag phase. The degree of saturation of PDC with Mg(2+)-thiamine pyrophosphate appears to have a central role in controlling the lag phase and the degree of cooperativity.

Journal Article↗
PubMed2004

Pyruvate decarboxylase, the target for omeprazole in metronidazole-resistant and iron-restricted Tritrichomonas foetus.

The substituted benzimidazole omeprazole, used for the treatment of human peptic ulcer disease, inhibits the growth of the metronidazole-resistant bovine pathogen Tritrichomonas foetus in vitro (MIC at which the growth of parasite cultures is inhibited by 50%, 22 microg/ml [63 microM]). The antitrichomonad activity appears to be due to the inhibition of pyruvate decarboxylase (PDC), which is the key enzyme responsible for ethanol production and which is strongly upregulated in metronidazole-resistant trichomonads. PDC was purified to homogeneity from the cytosol of metronidazole-resistant strain. The tetrameric enzyme of 60-kDa subunits is inhibited by omeprazole (50% inhibitory concentration, 16 microg/ml). Metronidazole-susceptible T. foetus, which expresses very little PDC, is only slightly affected. Omeprazole has the same inhibitory effect on T. foetus cells grown under iron-limited conditions. Similarly to metronidazole-resistant cells, T. foetus cells grown under iron-limited conditions have nonfunctional hydrogenosomal metabolism and rely on cytosolic PDC-mediated ethanol fermentation.

Animals↗
PubMed2003

Contrasting effects of submergence in light and dark on pyruvate decarboxylase activity in roots of rice lines differing in submergence tolerance.

Submergence tolerance of 13 doubled haploid lines of rice and their parents (submergence tolerant FR13A and submergence intolerant CT6241) was assessed using 2-week-old seedlings. Plants were scored for leaf senescence and percentage of seedlings that survived up to 15 d submergence, followed by a 12 d recovery period. Seven lines proved to be submergence tolerant, and six relatively intolerant. In all lines, activity of pyruvate decarboxylase (PDC), extracted from the apical 3-5 cm of root axes, decreased by 46-96 % and 38-76 %, respectively, during 5 or 10 d submergence under natural day/night conditions, compared with pre-submergence values (100 %). However, when the enzyme was extracted at night, submergence increased PDC activity of all rice lines (approx. 112 % on average), compared with pre-submergence values (100 %). The stimulating effect of the dark period on PDC activity was reproduced and amplified by submerging rice seedlings for up to 5 d in continuous darkness in water containing sub-ambient concentrations of oxygen (2.3 mg l(-1)). Such increased PDC activity was also observed in seedlings exposed to anoxia for 6 h (approx. 6-175 % higher than pre-submergence values). Irrespective of tolerance class, submergence decreased soluble protein concentrations under all conditions and sampling times. No positive correlation was found between PDC activity and tolerance of the various rice lines to submergence. However, PDC activity was slightly higher in submergence intolerant lines, compared with tolerant lines, under both dark submergence and anoxia. Such differences in PDC activity between the two groups of rice lines were not observed when they were submerged under the natural diurnal cycle. Increased PDC activity in roots at night demonstrated a probable incidence of tissue hypoxia or anoxia during submergence during each dark period.

Adaptation, Physiological↗
PubMed2003

Histone H1 Is required for proper regulation of pyruvate decarboxylase gene expression in Neurospora crassa.

We show that Neurospora crassa has a single histone H1 gene, hH1, which encodes a typical linker histone with highly basic N- and C-terminal tails and a central globular domain. A green fluorescent protein-tagged histone H1 chimeric protein was localized exclusively to nuclei. Mutation of hH1 by repeat-induced point mutation (RIP) did not result in detectable defects in morphology, DNA methylation, mutagen sensitivity, DNA repair, fertility, RIP, chromosome pairing, or chromosome segregation. Nevertheless, hH1 mutants had mycelial elongation rates that were lower than normal on all tested carbon sources. This slow linear growth phenotype, however, was less evident on medium containing ethanol. The pyruvate decarboxylase gene, cfp, was abnormally derepressed in hH1 mutants on ethanol-containing medium. This derepression was also found when an ectopically integrated fusion of the cfp gene promoter to the reporter gene hph was analyzed. Thus, Neurospora histone H1 is required for the proper regulation of cfp, a gene with a key role in the respiratory-fermentative pathway.

Amino Acid Sequence↗
PubMed2005

Transcriptional analysis of the Azospirillum brasilense indole-3-pyruvate decarboxylase gene and identification of a cis-acting sequence involved in auxin responsive expression.

Expression of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase, a key enzyme in the production of indole-3-acetic acid (IAA) in this bacterium, is upregulated by IAA. Here, we demonstrate that the ipdC gene is the promoter proximal gene in a bicistronic operon. Database searches revealed that the second gene of this operon, named iaaC, is well conserved evolutionarily and that the encoded protein is homologous to the Escherichia coli protein SCRP-27A, the zebrafish protein ES1, and the human protein KNP-I/GT335 (HES1), all of unknown function and belonging to the DJ-1/PfpI superfamily. In addition to this operon structure, iaaC is also transcribed monocistronically. Mutation analysis of the latter gene indicated that the encoded protein is involved in controlling IAA biosynthesis but not ipdC expression. Besides being upregulated by IAA, expression of the ipdC-iaaC operon is pH dependent and maximal at acidic pH. The ipdC promoter was studied using a combination of deletion analyses and site-directed mutagenesis. A dyadic sequence (ATTGTTTC(GAAT)GAAACAAT), centered at -48 was demonstrated to be responsible for the IAA inducibility. This bacterial auxin-responsive element does not control the pH-dependent expression of ipdC-iaaC.

5' Flanking Region↗
PubMed1998

Identification and isolation of the indole-3-pyruvate decarboxylase gene from Azospirillum brasilense Sp7: sequencing and functional analysis of the gene locus.

The root-associated bacterium Azospirillum brasilense Sp7 produces the growth-stimulating phytohormone indole-3-acetic acid (= IAA) via the indole-3-pyruvate pathway. The DNA region containing ipdC, the structural gene for indole-3-pyruvate decarboxylase, was identified in a cosmid gene library of strain Sp7 by hybridization and has been sequenced. Upstream of the gene, two other ORF homologous to gltX and cysS were sequenced that are transcribed in the opposite direction. A functional analysis of the cloned ipdC region has been performed. To test the expression of the gene, a lacZ-Km cartridge was introduced into the gene. By this construct, tryptophan-dependent stimulation of gene expression in A. brasilense Sp7 was observed. Evidences for the existence of another copy of the ipdC gene in the Azospirillum genome are also reported.

Azospirillum brasilense↗