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Results for “Pyruvate Decarboxylase”

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PubMed1992

Resolution of brewer's yeast pyruvate decarboxylase into multiple isoforms with similar subunit structure and activity using high-performance liquid chromatography.

A method for the purification of brewer's yeast pyruvate decarboxylase (EC 4.1.1.1) that resolves the enzyme into multiple active isoforms was developed. Seven activity fractions are resolved by DEAE HPLC chromatography. Among these fractions, three distinct subunit composition isoforms are apparent by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: alpha 4, a homotetrameric holoenzyme consisting of the lower mass subunit; alpha 2 beta 2, a heterotetrameric holoenzyme consisting of lower and higher mass subunits; and beta 4, a homotetrameric holoenzyme consisting of the higher mass subunit. Beta 4 is a heretofore unreported form which may represent the unproteolyzed form of the enzyme. The Km and Vmax for the alpha 4 and beta 4 isoforms are identical within the limits of experimental error, as is their behavior vis-à-vis the allosteric regulator pyruvamide. All active isoforms exist as tetramers according to gel filtration analysis under native conditions. The purification has been successfully applied to pyruvate decarboxylase isolated from two different species of yeast and therefore is likely to be of general utility for purification of this enzyme from other yeast sources. Conditions under which all three isoforms demonstrate exceptional stability, making them amenable to prolonged physicochemical studies at 4 degrees C and even at room temperature are reported.

Allosteric Regulation↗

PubMed1979

Irreversible inactivation of pyruvate decarboxylase in the presence of substrate and an oxidant. An example of paracatalytic enzyme inactivation.

Pyruvate decarboxylase from yeast is progressively inactivated in the presence of pyruvate and an extrinsic oxidant such as 2,6-dichloroindophenol or hexacyanoferrate(III). The inactivation is linked to the oxidation of the hydroxyethylthiamine diphosphate intermediate to acetate. Removal of low-molecular compounds by gel filtration does not reactivate the enzyme. The rate of inactivation obeys saturation kinetics with respect to substrate concentration and is independent of enzyme concentration. In analogy to the paracatalytic inactivation of other enzymes forming oxidizable carbanion intermediates [Christen, P. (1977) Methods Enzymol.46, 48--54], the oxidation of enzyme-bound hydroxyethylthiamine diphosphate is thought to generate a transiently reactive intermediate which, without being released from the enzyme, covalently modifies a group at or near the active site. Reconstitution experiments indicate that the protein rather than the coenzyme moiety is modified.

2,6-Dichloroindophenol↗

PubMed1994

Molecular cloning and sequence analysis of an Azospirillum brasilense indole-3-pyruvate decarboxylase gene.

Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading frame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.

Amino Acid Sequence↗

PubMed1991

Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II.

Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high level of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).

Acetyltransferases↗

PubMed1999

The linkage of catalysis and regulation in enzyme action: oxidative diversion in the hysteretically regulated yeast pyruvate decarboxylase.

The reaction catalyzed by the thiamin-diphosphate-dependent yeast pyruvate decarboxylase, which is hysteretically regulated by pyruvate, undergoes paracatalytic oxidative diversion by 2,6-dichlorophenolindophenol, which traps a carbanionic intermediate and diverts the product from acetaldehyde to acetate (Christen, P. Meth. Enzymol. 1977, 46, 48). This reaction is now shown to exhibit an oxidant on-rate constant somewhat faster than that for pyruvate in the normal catalytic cycle and a product off-rate constant about 60-fold smaller than that for acetaldehyde. Both on-rates and off-rates exhibit an inverse solvent isotope effect of 1.5-2, observed in normal catalysis as a signal of sulfhydryl addition to the keto group of pyruvate at the allosteric regulatory site. The findings are consistent with a model for regulation in which the sulfhydryl-addition process mediates access to a fully catalytically competent active site, the oxidative-diversion reaction being forced to make use of the normal entry exit machinery.

2,6-Dichloroindophenol↗

PubMed1983

The synthesis of yeast pyruvate decarboxylase is regulated by large variations in the messenger RNA level.

The yeast PDC1 gene coding for the fermentative enzyme pyruvate decarboxylase was isolated. This DNA sequence was used to identify the corresponding messenger RNA by hybridization. It could be shown that the synthesis of pyruvate decarboxylase is efficiently regulated by variations in the amount of PDC1 mRNA. Very low levels of PDC1 mRNA were found in cells growing in a medium containing ethanol. Glucose addition to these cells leads to a rapid accumulation of PDC1 mRNA. The PDC1 mRNA levels found in different mutants and in cells growing in media containing carbon sources other than glucose or ethanol suggest that the amount of PDC1 mRNA in yeast cells is affected by a number of different factors.

Carboxy-Lyases↗

PubMed2000

Mutagenesis at asp27 of pyruvate decarboxylase from Zymomonas mobilis. Effect on its ability to form acetoin and acetolactate.

Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a bivalent cation as essential cofactors. The three-dimensional structure of PDC from Zymomonas mobilis (ZMPDC) shows that Asp27 (D27) is close to ThDP in the active site, and mutagenesis of this residue has suggested that it participates in catalysis. The normal product of the PDC reaction is acetaldehyde but it is known that the enzyme can also form acetoin as a by-product from the hydroxyethyl-ThDP reaction intermediate. This study focuses on the role of D27 in the production of acetoin and a second by-product, acetolactate. D27 in ZMPDC was altered to alanine (D27A) and this mutated protein, the wild-type, and two other previously constructed PDC mutants (D27E and D27N) were expressed and purified. Determination of the kinetic properties of D27A showed that the affinity of D27A for ThDP is decreased 30-fold, while the affinity for Mg2+ and the Michaelis constant for pyruvate were similar to those of the wild-type. The time-courses of their reactions were investigated. Each mutant has greatly reduced ability to produce acetaldehyde and acetoin compared with the wild-type PDC. However, the effect of these mutations on acetaldehyde production is greater than that on acetoin formation. The D27A mutant can also form acetolactate, whereas neither of the other mutants, nor the wild-type PDC, can do so. In addition, acetaldehyde formation and/or release are reversible in wild-type ZMPDC but irreversible for the mutants. The results are explained by a mechanism involving thermodynamic and geometric characteristics of the intermediates in the reaction.

Acetaldehyde↗

PubMed1984

Interaction of thiamin diphosphate and thiamin thiazolone diphosphate with wheat germ pyruvate decarboxylase.

The interactions of the apoenzyme of wheat germ pyruvate decarboxylase with thiamin diphosphate and with thiamin thiazolone diphosphate have been investigated. The results test hypotheses concerning the structure of the transition state for decarboxylation of the enzyme-bound adduct of pyruvate and thiamin diphosphate. Thiamin thiazolone diphosphate, a possible transition state analogue, binds to the apoenzyme by a two-step process. The first is slow and reversible (k = 200 M-1 s-1; K = 5 X 10(-7) M). The second step is irreversible (k = 1 X 10(-6) s-1). The rate constant for activation by thiamin diphosphate is 160 M-1 s-1. Thiamin diphosphate is released very slowly from the holoenzyme (k = 2 X 10(-5) s-1). Thiamin thiazolone diphosphate competitively inhibits activation of the apoenzyme by thiamin diphosphate, Ki = 2 X 10(-6) M. Km for thiamin diphosphate is only 3 times larger. Thiamin thiazolone is solvated preferentially to thiamin in 2-butanol, a medium whose polarity should resemble that of the binding site. It is concluded that the observed high affinity of thiamin thiazolone diphosphate for the apoenzyme is the result of a combination of effects which do not require the assumption that it is an analogue of the transition state for the decarboxylation of enzyme-bound 2-(2-lactyl) thiamin diphosphate.

Carboxy-Lyases↗

PubMed2001

Production of the Gram-positive Sarcina ventriculi pyruvate decarboxylase in Escherichia coli.

Sarcina ventriculi grows in a remarkable range of mesophilic environments from pH 2 to pH 10. During growth in acidic environments, where acetate is toxic, expression of pyruvate decarboxylase (PDC) serves to direct the flow of pyruvate into ethanol during fermentation. PDC is rare in bacteria and absent in animals, although it is widely distributed in the plant kingdom. The pdc gene from S. ventriculi is the first to be cloned and characterized from a Gram-positive bacterium. In Escherichia coli, the recombinant pdc gene from S. ventriculi was poorly expressed due to differences in codon usage that are typical of low-G+C organisms. Expression was improved by the addition of supplemental codon genes and this facilitated the 136-fold purification of the recombinant enzyme as a homo-tetramer of 58 kDa subunits. Unlike Zymomonas mobilis PDC, which exhibits Michaelis-Menten kinetics, S. ventriculi PDC is activated by pyruvate and exhibits sigmoidal kinetics similar to fungal and higher plant PDCs. Amino acid residues involved in the allosteric site for pyruvate in fungal PDCs were conserved in S. ventriculi PDC, consistent with a conservation of mechanism. Cluster analysis of deduced amino acid sequences confirmed that S. ventriculi PDC is quite distant from Z. mobilis PDC and plant PDCs. S. ventriculi PDC appears to have diverged very early from a common ancestor which included most fungal PDCs and eubacterial indole-3-pyruvate decarboxylases. These results suggest that the S. ventriculi pdc gene is quite ancient in origin, in contrast to the Z. mobilis pdc, which may have originated by horizontal transfer from higher plants.

Amino Acid Sequence↗

PubMed1989

Maize pyruvate decarboxylase mRNA is induced anaerobically.

A cDNA was identified using an oligonucleotide designed by comparing the sequences of bacterial and yeast pyruvate decarboxylase. The sequence of the cDNA identified by the oligonucleotide contained an open reading frame that encoded a protein of 65 kDa that was similar in sequence to bacterial and yeast pyruvate decarboxylase. This protein was selectively precipitated by an antiserum specific for maize PDC. Northern-blot analysis shows that PDC mRNA is anaerobically induced. Southern-blot analysis of maize genomic DNA indicated that the maize PDC gene has a single or low copy number.

Amino Acid Sequence↗

PubMed1979

[Small angle X-ray scattering studies on the structure and symmetry of yeast pyruvate decarboxylase in solution].

The holo-enzyme of the pyruvate decarboxylase EC 4.1.1.1 isolated from brewer's yeast was studied by small-angle X-ray scattering. The point-group symmetry 222 deduced from electron microscopic investigations was confirmed for molecules in solution. The best approximation of the overall shape of the enzyme was reached by an homogeneous triaxial ellipsoid, with the half-axes of a = 2.47 nm, b = 5.08 nm and c = 8.0 nm. The ellipsoid is halved vertical to the a-axis, and both halves are turned from each other by about 30 degrees. As an explanation of the existence of four binding sites for Mg2+ and thiamine pyrophosphate on the dimeric molecule the hypothesis of a gene duplication is discussed.

Binding Sites↗

PubMed1995

The replacement of Trp392 by alanine influences the decarboxylase/carboligase activity and stability of pyruvate decarboxylase from Zymomonas mobilis.

The bulky tryptophan residue 392 located in the deep cleft leading to the active center of pyruvate decarboxylase (PDC) from Zymomonas mobilis was changed to alanine which is found in the equivalent position of PDC from yeast. The mutation reduced the decarboxylase activity towards pyruvate by a factor of two (60-70 U/mg), whereas the Km (1.1 mM in Mes/KOH buffer) remains unchanged compared with the wild-type enzyme. The apparent Km for thiamine diphosphate (thiamin-P2) in the presence of 5 mM MgSO4 was increased by a factor of 10 (84 microM in Mes/KOH buffer) and the tetrameric mutant protein was less stable, as indicated by urea denaturation experiments. The mutation enhanced the carboligase activity of the enzyme towards benzaldehyde by a factor of four. The resulting alpha-hydroxyketone was identified as (R)-phenylacetylcarbinol.

Acetone↗

PubMed1996

Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose.

In Saccharomyces cerevisiae, the structural genes PDC1, PDC5 and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild-type strain, using the dominant marker genes APT1 and Tn5ble. A pyruvate-decarboxylase-negative (Pdc-) mutant lacking all three PDC genes exhibited a three-fold lower growth rate in complex medium with glucose than the isogenic wild-type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc- strains. Furthermore, in ethanol-limited chemostat cultures, the biomass yield of Pdc- and wild-type S. cerevisiae were identical. However, Pdc- S. cerevisiae was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol-limited chemostat cultures (D = 0 center dot 10 h-1) were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed-substrate cultures were switched to a feed containing glucose as the sole carbon source, wash-out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl-CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose-limited chemostat cultures.

Culture Media↗

PubMed1978

The mechanism of substrate activation of pyruvate decarboxylase: a first approach.

The sigmoidal shape of the curve for v[S], characteristic of pyruvate decarboxylase, indicates that the catalytic activity of this enzyme is regulated by the substrate. The enzyme, which is inactive in the absence of its substrate, is activated not only by 2-oxo acids but also by 2-oxo acid amides, which cannot act as a substrate of the enzyme. Whilst the dissociation constant of the enzyme-activator complex depends on the electrophilic nature of the carbonyl group, the catalytic activity reached at saturation concentrations of the activator species is independent of the structure of the activator molecules. The mechanism of activation which proceeds via two reversible steps could be evaluated exactly by stopped-flow techniques. The kinetic parameters of the activation and deactivation reaction were estimated and the validity of the equations derived which describe the activation kinetics could be proved by comparing them with the measured data. Using glyoxylic acid as an irreversibly binding active-site marker, it could be shown that addition of the substrate to the enzyme-bound thiamin diphosphate is the step of the catalytic mechanism whose rate is controlled by the substrate (activator) molecule.

Carboxy-Lyases↗

PubMed1995

Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium.

Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) exhibits biphasic kinetics with 52 +/- 11% of the PDC reacting with k1 = (1.0 +/- 0.3) x 10(-2) s-1 and 48 +/- 12% of the PDC reacting with k2 = (1.1 +/- 0.6) x 10(-1) s-1 when TDP (KTDP = 0.5 +/- 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 +/- 0.5) x 10(-2) s-1 upon addition of Mg2+ (KMg2+ = 0.2 +/- 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 < or = 3-fold and has no effect on the value of k1.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoenzymes↗

PubMed2001

Consequences of a modified putative substrate-activation site on catalysis by yeast pyruvate decarboxylase.

Earlier, it had been proposed in the laboratories at Halle that a cysteine residue is responsible for the hysteretic substrate activation behavior of yeast pyruvate decarboxylase. More recently, this idea has received support in a series of studies from Rutgers with the identification of residue C221 as the site where substrate is bound to transmit the information to H92, to E91, to W412, and finally to the active center thiamin diphosphate. According to steady-state kinetic assays, the C221A/C222A variant is no longer subject to substrate activation yet is still a well-functioning enzyme. Several further experiments are reported on this variant: (1) The variant exhibits lag phases in the product formation progress curves, which can be attributed to a unimolecular step in the pre-steady-state stage of catalysis. (2) The rate of exchange with solvent deuterium of the thiamin diphosphate C2H atom is slowed by a factor of 2 compared to the wild-type enzyme, suggesting that the reduced activity that results from the substitutions some 20 A from the active center is also seen in the first key step of the reaction. (3) The solvent (deuterium oxide) kinetic isotope effect was found to be inverse on V(max)/K(m) (0.62), and small but normal on V(max) (1.26), virtually ruling out residue C221 as being responsible for the inverse effects reported for the wild-type enzyme at low substrate concentrations. The solvent kinetic isotope effects are compared to those on two related enzymes not subject to substrate activation, Zymomonas mobilis pyruvate decarboxylase and benzoylformate decarboxylase.

Alanine↗

PubMed1993

Transient improvement of congenital lactic acidosis in a male infant with pyruvate decarboxylase deficiency treated with dichloroacetate.

A comatose male newborn infant with congenital lactic acidosis caused by pyruvate decarboxylase deficiency was treated with dichloroacetate (DCA), which stimulated an 88% drop in serum lactate concentration and reversed his coma. The response to DCA was temporary and the lactic acidosis worsened until his death, but DCA may confer more lasting benefit in less severely affected infants.

Acidosis, Lactic↗

PubMed1994

The nucleotide sequence and initial characterization of pyruvate decarboxylase from the yeast Hanseniaspora uvarum.

We have isolated a pyruvate decarboxylase (PDC) gene from the yeast Hanseniaspora uvarum using the Saccharomyces cerevisiae PDC1 gene as a probe. The nucleotide sequence of this gene was determined and compared to PDC genes from yeast and other organisms. The H. uvarum PDC gene is more than 70% identical to the S. cerevisiae PDC isozymes and possesses a putative thiamine diphosphate binding site. The PDC enzyme was purified and partially characterized. The H. uvarum PDC was very similar to other known PDCs; the Km for pyruvate was 0.75 mM, and the enzyme is a homotetramer with subunits of M(r) = 57,000.

Amino Acid Sequence↗