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At least 19 recordsLinked to original sources
PubMed1997

Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase.

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C by fermenting carbohydrates and peptides. The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 and reduces P. furiosus ferredoxin. Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction. Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role. Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production. The apparent Km values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90 degrees C. These data are comparable to those previously determined for the pyruvate oxidation reaction of POR. At 80 degrees C (pH 8.0), the apparent Vm value for pyruvate decarboxylation was about 40% of the apparent Vm value for pyruvate oxidation rate (using P. furiosus ferredoxin as the electron acceptor). Tentative catalytic mechanisms for these two reactions are presented. In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea. It is proposed that the various aldehydes produced by these oxidoreductases in vivo are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown.

Aldehyde Oxidoreductases↗
PubMed1993

The relationships between transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase of the pyruvate dehydrogenase complex.

The amino acid sequences of four thiamine pyrophosphate-requiring enzymes were aligned with the published amino acid sequence of the transketolase of Hansenula polymorpha. Sequences of the combined alpha and beta subunits of the E1 enzyme of the pyruvate dehydrogenase complexes of Homo sapiens and Bacillus stearothermophilus aligned well with the transketolase while the E1 of the pyruvate dehydrogenase complex of Escherichia coli aligned easily provided a non-aligning segment of 77 amino acids was omitted. The non-acetylating pyruvate decarboxylase of Saccharomyces cerevisiae could only be aligned if the sequence was cut in two with the C-terminus corresponding to the N-terminus of the other TPP-dependent enzymes. Using the published 2.5 A resolution of the X-ray crystal structure of Saccharomyces cerevisiae transketolase as a template we show that a hydrophobic region of the beta-subunit of the PDH E1 alpha beta enzymes likely contains a binding site for the thiazolium ring of TPP and key motifs are retained in common by all the TPP-dependent enzymes considered, which are essential for catalysis.

Amino Acid Sequence↗
PubMed1999

Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity.

Pyruvate decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of pyruvate decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of pyruvate-decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN. PK113-7D. This caused a 3-4-fold reduction of pyruvate-decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h(-1). Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2Delta strain, formation of ethanol and acetate was reduced by 60-70%. In contrast to the wild type, the pdc2Delta strain produced substantial amounts of pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2Delta cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced pyruvate-decarboxylase activities, glycolytic flux is controlled by NADH reoxidation. In aerobic, glucose-limited chemostat cultures, the wild type exhibited a mixed respiro-fermentative metabolism at dilution rates above 0.30 h(-1). Below this dilution rate, sugar metabolism was respiratory. At dilution rates up to 0.20 h(-1), growth of the pdc2Delta strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low micro(max) of the pdc2Delta strain in glucose-limited chemostat cultures indicates that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for pyruvate. Furthermore, it implies that inactivation of PDC2 is not a viable option for reducing byproduct formation in industrial fermentations.

Acetates↗
PubMed1998

High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis. Implications for substrate activation in pyruvate decarboxylases.

The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 A resolution and refined to a crystallographic R-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the alpha/beta type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.

Amino Acid Sequence↗
PubMed1982

Effect of pyruvate decarboxylase activity and of pyruvate concentration on the production of 1-hydroxy-1-phenylpropanone in Saccharomyces carlsbergensis.

The assumption that the pyruvate decarboxylase activity of Saccharomyces carlsbergensis is the main limiting factor determining the formation rate and the total amount of d(--)-l-hydroxy-l-phenyl-propanone (phenylacetylcarbinol, PAC) produced was not confirmed. An increase of about 30% of the total amount of the PAC produced was obtained when 8.5% sodium pyruvate was gradually added. The total PAC production is probably influenced both by the pyruvate decarboxylase activity and the pyruvate concentration in the cells, the latter being actually the determining rate-limiting factor.

Acetone↗
PubMed2000

Sites of limited proteolysis in the pyruvate decarboxylase component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus and their role in catalysis.

The E1 component (pyruvate decarboxylase) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus is a heterotetramer (alpha2beta2) of E1alpha and E1beta polypeptide chains. The domain structure of the E1alpha and E1beta chains, and the protein-protein interactions involved in assembly, have been studied by means of limited proteolysis. It appears that there may be two conformers of E1alpha in the E1 heterotetramer, one being more susceptible to proteolysis than the other. A highly conserved region in E1alpha, part of a surface loop at the entrance to the active site, is the most susceptible to cleavage in E1 (alpha2beta2). As a result, the oxidative decarboxylation of pyruvate catalysed by E1 in the presence of dichlorophenol indophenol as an artificial electron acceptor is markedly enhanced, but the reductive acetylation of a free lipoyl domain is unchanged. The parameters of the interaction between cleaved E1 and the peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase E2 component are identical to those of the wild-type E1. However, a pyruvate dehydrogenase complex assembled in vitro with cleaved E1p exhibits a markedly lower overall catalytic activity than that assembled with untreated E1. This implies that active site coupling between the E1 and E2 components has been impaired. This has important implications for the way in which a tethered lipoyl domain can interact with E1 in the assembled complex.

Amino Acid Sequence↗
PubMed1991

Characterization of PDC6, a third structural gene for pyruvate decarboxylase in Saccharomyces cerevisiae.

Pyruvate decarboxylase is the key enzyme in alcoholic fermentation in yeast. Two structural genes, PDC1 and PDC5 have been characterized. Deletion of either of these genes has little or no effect on the specific pyruvate decarboxylase activity, but enzyme activity is undetectable in mutants lacking both PDC1 and PDC5 (S. Hohmann and H. Cederberg, Eur. J. Biochem. 188:615-621, 1990). Here I describe PDC6, a gene structurally closely related to PDC1 and PDC5. The product of PDC6 does not seem to be required for wild-type pyruvate decarboxylase activity in glucose medium; delta pdc6 mutants have no reduced specific enzyme activity, and the PDC6 deletion did not change the phenotype or the specific enzyme activity of mutants lacking either or both of the other two structural genes. However, in cells grown in ethanol medium the PDC6 deletion caused a reduction of pyruvate decarboxylase activity. Northern (RNA) blot analysis showed that PDC6 is weakly expressed, and expression seemed to be higher during growth in ethanol medium. This behavior remained obscure since pyruvate decarboxylase catalyzes an irreversible reaction. Characterization of all combinations of PDC structural gene deletion mutants, which produce different amounts of pyruvate decarboxylase activity, showed that the enzyme is also needed for normal growth in galactose and ethanol medium and in particular for proper growth initiation of spores germinating on ethanol medium.

Amino Acid Sequence↗
PubMed1990

Inhibition of pyruvate:ferredoxin oxidoreductase from Trichomonas vaginalis by pyruvate and its analogues. Comparison with the pyruvate decarboxylase component of the pyruvate dehydrogenase complex.

Pyruvate:ferredoxin oxidoreductase and the pyruvate dehydrogenase multi-enzyme complex both catalyse the CoA-dependent oxidative decarboxylation of pyruvate but differ in size, subunit composition and mechanism. Comparison of the pyruvate:ferredoxin oxidoreductase from the protozoon Trichomonas vaginalis and the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex shows that both are inactivated by incubation with pyruvate under aerobic conditions in the absence of co-substrates. However, only the former is irreversibly inhibited by incubation with hydroxypyruvate, and only the latter by incubation with bromopyruvate. Pyruvate:ferredoxin oxidoreductase activity is potently, but reversibly, inhibited by addition of bromopyruvate in the presence of CoA, and it is suggested that the mechanism involves formation of an adduct between CoA and bromopyruvate in the active site of the enzyme. It is proposed that both enzymes are inactivated by pyruvate through a mechanism involving oxidation of an enzyme-bound thiamin pyrophosphate/substrate adduct to form a tightly bound inhibitory species, possibly thiamin thiazolone pyrophosphate as hypothesized by Sumegi & Alkonyi.

Animals↗
PubMed1992

Purification and characterization of pyruvate decarboxylase from Sarcina ventriculi.

Pyruvate decarboxylase from the obligate anaerobe Sarcina ventriculi was purified eightfold. The subunit Mr was 57,000 +/- 3000 as estimated from SDS-PAGE, and the native Mr estimated by gel filtration on a Superose 6 column was 240,000, indicating that the enzyme is a tetramer. The Mr values are comparable to those for pyruvate decarboxylase from Zymomonas mobilis and Saccharomyces cerevisiae, which are also tetrameric enzymes. The enzyme was oxygen stable, and had a pH optimum within the range 6.3-6.7. It displayed sigmoidal kinetics for pyruvate, with a S0.5 of 13 mM, kinetic properties also found for pyruvate decarboxylase from yeast and differing from the Michaelis-Menten kinetics of the enzyme from Z. mobilis. No activators were found. p-Chloromercuribenzoate inhibited activity and the inhibition was reversed by the addition of dithiothreitol, indicating that cysteine is important in the active site. The N-terminal amino acid sequence of pyruvate decarboxylase was more similar to the sequence of S. cerevisiae than Z. mobilis pyruvate decarboxylase.

Amino Acid Sequence↗
PubMed1999

Aspartate-27 and glutamate-473 are involved in catalysis by Zymomonas mobilis pyruvate decarboxylase.

Zymomonas mobilis pyruvate decarboxylase (EC 4.1.1.1) was subjected to site-directed mutagenesis at two acidic residues near the thiamin diphosphate cofactor in the active site. Asp-27 was changed to Glu or Asn, and Glu-473 was mutated to Asp (E473D) or Gln (E473Q). Each mutant protein was purified to near-homogeneity, and the kinetic and cofactor-binding properties were compared with those of the wild-type protein. Despite the very conservative nature of these alterations, all mutants had a very low, but measurable, specific activity ranging from 0.025% (E473Q) to 0.173% (E473D) of the wild type. With the exception of E473Q, the mutants showed small decreases in the affinity for thiamin diphosphate, and binding of the second cofactor (Mg2+) was also weakened somewhat. With E473Q, both cofactors seemed to be very tightly bound so that they were not removed by the treatment that was effective for the wild-type enzyme and other mutant forms. All mutants showed minor changes in the Km for substrate, but these alterations did not account for the low activities. These low specific activities, accompanied by little change in the Km for pyruvate, are consistent with a quantitative model of the catalytic cycle in which the main effect of the mutations is to slow the decarboxylation step with a minor change in the rate constant for pyruvate binding.

Aspartic Acid↗
PubMed2003

Induction of Rhizopus oryzae pyruvate decarboxylase genes.

Two pyruvate decarboxylase genes, pdcA, and pdcB, were cloned from Rhizopus oryzae. These genes are similar to each other with approximately 85% nucleotide sequence identity within the coding region. Multiple transcriptional start sites and polyadenylation sites were found for both genes. The deduced translation product of each gene results in a 561 amino acid protein with approximate molecular weight of 61 kDa each. The amino acid identity between the two proteins was 91% as calculated by Lipmann-Pearson comparisons. Transcriptional control appears to be important in regulation of the PDC, since much of the transcript accumulation parallels enzymatic activity. There was no detectable pdc transcript from cultures grown in glycerol-containing medium. Induction of transcription for pdcA and pdcB was initiated within 1.5 h of adding glucose to the culture. Shifting the aerobically grown cultures to anoxic conditions at this time resulted in enhanced pdc transcription, PDC enzymatic activity, and ethanol production, compared to cultures with continued aerobic growth.

Cloning, Molecular↗
PubMed1990

Inactivation of pyruvate decarboxylase by 3-hydroxypyruvate.

Pyruvate decarboxylase from Zymomonas mobilis is inhibited by 3-hydroxypyruvate, which can also act as a poor substrate. While catalysing the decarboxylation of this alternative substrate, the enzyme undergoes a progressive but partial inactivation over several hours. The extent of inactivation depends upon the pH and upon the concentration of 3-hydroxypyruvate. After partial inactivation and removal of unchanged 3-hydroxypyruvate, enzymic activity recovers slowly. We suggest that inactivation results from accumulation of enzyme-bound glycollaldehyde, which is relatively stable, possibly because it is dehydrated to form an acetyl group.

Binding, Competitive↗
PubMed1998

Effects of pyruvate decarboxylase overproduction on flux distribution at the pyruvate branch point in Saccharomyces cerevisiae.

A multicopy plasmid carrying the PDC1 gene (encoding pyruvate decarboxylase; Pdc) was introduced in Saccharomyces cerevisiae CEN. PK113-5D. The physiology of the resulting prototrophic strain was compared with that of the isogenic prototrophic strain CEN.PK113-7D and an empty-vector reference strain. In glucose-grown shake-flask cultures, the introduction of the PDC1 plasmid caused a threefold increase in the Pdc level. In aerobic glucose-limited chemostat cultures growing at a dilution rate of 0.10 h-1, Pdc levels in the overproducing strain were 14-fold higher than those in the reference strains. Levels of glycolytic enzymes decreased by ca. 15%, probably due to dilution by the overproduced Pdc protein. In chemostat cultures, the extent of Pdc overproduction decreased with increasing dilution rate. The high degree of overproduction of Pdc at low dilution rates did not affect the biomass yield. The dilution rate at which aerobic fermentation set in decreased from 0.30 h-1 in the reference strains to 0.23 h-1 in the Pdc-overproducing strain. In the latter strain, the specific respiration rate reached a maximum above the dilution rate at which aerobic fermentation first occurred. This result indicates that a limited respiratory capacity was not responsible for the onset of aerobic fermentation in the Pdc-overproducing strain. Rather, the results indicate that Pdc overproduction affected flux distribution at the pyruvate branch point by influencing competition for pyruvate between Pdc and the mitochondrial pyruvate dehydrogenase complex. In respiratory cultures (dilution rate, <0.23 h-1), Pdc overproduction did not affect the maximum glycolytic capacity, as determined in anaerobic glucose-pulse experiments.

Aerobiosis↗
PubMedDate not supplied

[Paracatalytic inactivation of pyruvate decarboxylase in the presence of quinones].

Pyruvate promotes the yeast pyruvate decarboxylase inactivation under the influence of substituted p-benzoquinones. Pyruvate decarboxylase activity is not renewed after the removal of low-molecular impurities by gel filtration and subsequent addition of dithiothreitol, thiamine diphosphate, magnesium chloride. The inactivation rate under joint action of 2-methyl-5-isopropyl-p-benzoquinone and pyruvate is regulated by the pseudo-first-order equation. The relationship between pseudo-first-order rate constant and pyruvate concentration takes the shape of hyperbola. The inactivation order with respect to quinone is determined by oxidant concentration and pH value. Maximum pseudo-first-order rate constant values in the presence of the excess substrate and 2-methyl-5-isopropyl-p-benzoquinone are observed at pH 5.9-6.0. The data obtained evidence for the fact that during inactivation quinone interacts with "active acetaldehyde" being the intermediate in the process of catalysis with pyruvate decarboxylase.

Benzoquinones↗
PubMed1987

Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase.

DNA sequence analysis showed that pyruvate decarboxylase (one of the most abundant proteins in Zymomonas mobilis) contains 559 amino acids. The promoter for the gene encoding pyruvate decarboxylase was not recognized by Escherichia coli, although the cloned gene was expressed at relatively high levels under the control of alternative promoters. The promoter region did not contain sequences which could be identified as being homologous to the generalized promoter structure for E. coli. Hydropathy plots for the amino acid sequence indicated that pyruvate decarboxylase contains a large number of hydrophobic domains which may contribute to the thermal stability of this enzyme.

Amino Acid Sequence↗