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PubMed1987

Improved purification of pyruvate decarboxylase from wheat germ. Its partial characterisation and comparison with the yeast enzyme.

An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity-staining, produced two bands of different molecular masses and chain compositions. The high-molecular-mass band occurred in low quantity and consisted of, probably eight, apparently identical chains of Mr = 33,000, as judged from sodium dodecyl sulfate electrophoreses. The low-molecular-mass band contained two types of chains with Mr alpha = 63,000-65,000 and Mr beta = 61,000-62,000. The N termini of both chains were threonine, whereas their C-terminal sequences were different: alpha, -(Val)-(Ser)-(Ala)-Leu; beta, -(His)-(Asp)-(Ala)-Ser. Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different. In all properties investigated, the low-molecular-mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess (alpha beta)2 structure, the apoenzyme alpha beta. SH reagents inactivated the enzyme. Binding and fluorescence of 2-p-toluidinonaphthalene-6-sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2-Hydroxy-5-nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor-binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase.

Amino Acids↗
PubMed1983

Induction of pyruvate decarboxylase in Candida utilis.

Induction of cytoplasmic pyruvate decarboxylase (PDC) in Candida utilis increases with culture age throughout aerobic logarithmic phase growth on glucose and is due to oxygen depletion. Resting cells, grown on ethanol or acetate, fail to initiate PDC synthesis for two to three hours following anaerobic suspension in glucose although these cells are capable of glucose uptake. PDC induction occurs only at permissive growth temperatures, i.e., 5 degrees C to 35 degrees C. A preliminary characterization of the enzyme indicates an activity optimum of 35 degrees C and a lack of any regulatory properties.

Candida↗
PubMed1996

The 'petite-negative' yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity.

We cloned and sequenced the pyruvate decarboxylase (PDC; EC 4.1.1.1) structural gene KIPDCA in the yeast Kluyveromyces lactis and found it to be allelic to the previously isolated rag6 mutation. The putative amino acid sequence of the KIPdcAp appeared to be highly homologous to those of the yeast Pdc proteins identified so far. The disruption of KIPDCA indicated that it is the only PDC structural gene in K. lactis, as evidenced by the lack of PDC activity and ethanol production in the pdcA delta strains and by the absence of growth on glucose in the presence of respiratory inhibitors. It was observed that expression of the KIPDCA gene is induced by glucose at the transcriptional level. Transcription of the gene was reduced in the rag1, rag2, rag5 and rag8 mutants, which are defective for the low-affinity glucose permease, phosphoglucose isomerase, hexokinase, and a positive regulator of RAG1 expression, respectively.

Amino Acid Sequence↗
PubMed2001

Site-directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase: effect on activity and pH dependence.

Pyruvate decarboxylase (PDC, EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme about which there is a large body of structural and functional information. The active site contains several absolutely conserved ionizable groups and all of these appear to be important, as judged by the fact that mutation diminishes or abolishes catalytic activity. Previously we have shown [Schenk, G., Leeper, F.J., England, R., Nixon, P.F. & Duggleby, R.G. (1997) Eur. J. Biochem. 248, 63-71] that the activity is pH-dependent due to changes in kcat/Km while kcat itself is unaffected by pH. The effect on kcat/Km is determined by a group with a pKa of 6.45; the identity of this group has not been determined, although H113 is a possible candidate. Here we mutate five crucial residues in the active site with ionizable side-chains (D27, E50, H113, H114 and E473) in turn, to residues that are nonionizable or should have a substantially altered pKa. Each protein was purified and characterized kinetically. Unexpectedly, the pH-dependence of kcat/Km is largely unaffected in all mutants, ruling out the possibility that any of these five residues is responsible for the observed pKa of 6.45. We conjecture that the kcat/Km profile reflects the protonation of an alcoholate anion intermediate of the catalytic cycle.

Binding Sites↗
PubMed1981

Pyruvate decarboxylase deficiency in subacute necrotizing encephalomyelopathy.

A partial deficiency of pyruvate decarboxylase (PDC) was demonstrated in a child with hyperlactatemia and progressive ataxia, bulbar paresis, ophthalmoplegia, and polyneuropathy. Subacute necrotizing encephalomyelopathy (SNE) was found at necropsy. The association of SNE and PDC deficiency has been reported rarely, but a review of the diverse metabolic defects associated with SNE suggests that decreased PDC activity may be the common feature of SNE.

Alanine↗
PubMed1996

Crystal structure of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae at 2.3 A resolution.

The crystal structure of pyruvate decarboxylase (EC 4.1.1.1), a thiamin diphosphate-dependent enzyme isolated from Saccharomyces cerevisiae, has been determined and refined to a resolution of 2.3 A. Pyruvate decarboxylase is a homotetrameric enzyme which crystallizes with two subunits in an asymmetric unit. The structure has been refined by a combination of simulated annealing and restrained least squares to an R factor of 0.165 for 46,787 reflections. As in the corresponding enzyme from Saccharomyces uvarum, the homotetrameric holoenzyme assembly has approximate 222 symmetry. In addition to providing more accurate atomic parameters and certainty in the sequence assignments, the high resolution and extensive refinement resulted in the identification of several tightly bound water molecules in key structural positions. These water molecules have low temperature factors and make several hydrogen bonds with protein residues. There are six such water molecules in each cofactor binding site, and one of them is involved in coordination with the required magnesium ion. Another may be involved in the catalytic reaction mechanism. The refined model includes 1074 amino acid residues (two subunits), two thiamin diphosphate cofactors, two magnesium ions associated with cofactor binding and 440 water molecules. From the refined model we conclude that the resting state of the enzyme-cofactor complex is such that the cofactor is already deprotonated at the N4' position of the pyrimidine ring, and is poised to accept a proton from the C2 position of the thiazolium ring.

Amino Acid Sequence↗
PubMed1998

Structure and properties of pyruvate decarboxylase and site-directed mutagenesis of the Zymomonas mobilis enzyme.

Pyruvate decarboxylase (EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme that catalyzes the penultimate step in alcohol fermentation. The enzyme is widely distributed in plants and fungi but is rare in prokaryotes and absent in animals. Here we review its structure and properties with particular emphasis on how site-directed mutagenesis of the enzyme from Zymomonas mobilis has assisted us to understand the function of critical residues.

Amino Acid Sequence↗
PubMed2004

Purification, characterization, cloning and expression of pyruvate decarboxylase from Torulopsis glabrata IFO005.

In the production of pyruvate and optically active alpha-hydroxy ketones by Torulopsis glabrata, pyruvate decarboxylase (PDC, EC 4.1.1.1) plays an important role in pyruvate metabolism and in catalyzing the biotransformation of aromatic amino acid precursors to alpha-hydroxy ketones. In this paper, we have purified and characterized PDC from T. glabrata IFO005 and cloned the corresponding gene. A simple, rapid and efficient purification protocol was developed that provided PDC with high specific activity. Unlike other yeast or higher plant enzymes, known as homotetramers (alpha(4) or beta(4)) or heterotetramers (alpha(2)beta(2)), two active isoforms of PDC purified from T. glabrata IFO005 were homodimeric proteins with subunits of 58.7 kDa. We isolated the T. glabrata PDC gene encoding 563 amino acid residues and succeeded in overproducing the recombinant PDC protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Recombinant PDC from E. coli was purified as a homotetramer. Targeted gene disruption of PDC confirmed that T. glabrata has only one gene of PDC. This PDC gene showed about 80% homology with the genes of other yeasts, and amino acid residues involved in the allosteric site for pyruvate in other yeast PDCs were conserved in T. glabrata PDC. Both native PDC and recombinant PDC were activated by pyruvate and exhibited sigmoidal kinetics similar to those of Saccharomyces cerevisiae and higher plants. They also exhibited the similar catalytic properties: low thermostability, similar pH stability and optimal pH, and complete inhibition by glyoxylate.

Allosteric Site↗
PubMed1998

Transition-state theoretical interpretation of the catalytic power of pyruvate decarboxylases: the roles of static and dynamical considerations.

The catalytic power of two thiamin diphosphate (ThDP)-dependent enzymes, yeast pyruvate decarboxylase (the hysteretically regulated enzyme from Saccharomyces cerevisiae, SCPDC) and bacterial pyruvate decarboxylase (the unregulated enzyme from Zymomonas mobilis, ZMPDC), are analyzed by thorough-going application of transition-state theory, i.e. by a static approach that emphasizes the state-function character of the free energy of activation and takes no explicit account of dynamical considerations. The overall catalytic reaction is resolved into manifolds for addition (conversion of free enzyme and substrate to the complex of enzyme with the pyruvate:ThDP adduct), decarboxylation, and elimination (conversion of the complex of enzyme with the acetaldehyde:ThDP adduct formed by decarboxylation into free product and free enzyme). For SCPDC, the addition manifold is most strongly catalyzed (3x1012-fold, corresponding to net transition-state stabilization of 72 kJ/mol, transition-state stabilization of 83 kJ/mol diminished by reactant-state stabilization of 11 kJ/mol), the decarboxylation manifold is least strongly catalyzed (5x107-fold, corresponding to net transition-state stabilization of 41 kJ/mol, transition-state stabilization of 68 kJ/mol diminished by reactant-state stabilization of 27 kJ/mol), and the elimination manifold is catalyzed to an intermediate degree (2x1010-fold, corresponding to net transition-state stabilization of 59 kJ/mol, transition-state stabilization of 76 kJ/mol diminished by reactant-state stabilization of 17 kJ/mol). A similar situation holds for ZMPDC. There is no need to make an explicit analysis of dynamical factors in order to describe the catalytic mechanism and catalytic power of these complex enzymes.

Bacteria↗
PubMed1995

Purification and characterisation of pyruvate decarboxylase from pea seeds (Pisum sativum cv. Miko).

Pyruvate decarboxylase (PDC) was purified from pea seeds. The catalytically active holoenzyme is an oligomer of two types of subunits with molecular masses of about 65 kDa and 68 kDa, respectively. The active enzyme is a mixture of tetramers, octamers and even higher oligomers. These differences in the quaternary structure compared with PDC from yeast (tetramer) do not result in a different kinetic behaviour. The activity of pea PDC as well as that of yeast PDC is regulated by its substrate pyruvate resulting in a sigmoid shape of the v/S-plot. At the optimum pH of 6.0 a S0.5-value of 1 mM pyruvate is found that increases with rising pH and increasing concentrations of phosphate. The substrate analogue activator pyruvamide activates the enzyme resulting in a hyperbolic v/S-plot. The stability of PDC from pea seeds in solution is about one order of magnitude higher than that of yeast PDC. Despite the described similarities of the two enzymes no significant cross reactivity of the anti-pea PDC antibody with the enzyme from yeast occurs.

Blotting, Western↗
PubMed2002

Cloning and characterization of the Zymobacter palmae pyruvate decarboxylase gene (pdc) and comparison to bacterial homologues.

Pyruvate decarboxylase (PDC) is the key enzyme in all homo-ethanol fermentations. Although widely distributed among plants, yeasts, and fungi, PDC is absent in animals and rare in bacteria (established for only three organisms). Genes encoding the three known bacterial pdc genes have been previously described and expressed as active recombinant proteins. The pdc gene from Zymomonas mobilis has been used to engineer ethanol-producing biocatalysts for use in industry. In this paper, we describe a new bacterial pdc gene from Zymobacter palmae. The pattern of codon usage for this gene appears quite similar to that for Escherichia coli genes. In E. coli recombinants, the Z. palmae PDC represented approximately 1/3 of the soluble protein. Biochemical and kinetic properties of the Z. palmae enzyme were compared to purified PDCs from three other bacteria. Of the four bacterial PDCs, the Z. palmae enzyme exhibited the highest specific activity (130 U mg of protein(-1)) and the lowest Km for pyruvate (0.24 mM). Differences in biochemical properties, thermal stability, and codon usage may offer unique advantages for the development of new biocatalysts for fuel ethanol production.

Amino Acid Sequence↗
PubMed1975

Pyruvate decarboxylase III. Specificity restrictions for thiamine pyrophosphate in the protein association step, sub-unit structure.

Pyruvate decarboxylase dissociates into sub-units of one half the molecular weight at alkaline pH. At the same conditions the cofactors thiamine pyrophosphate and Mg2+ are released and can be separated from the protein. Thiamine pyrophosphate is an obligatory cofactor for reconstitution to the oligomer [1]. In this study the effect of thiamine pyrophosphate derivatives (thiamine monophosphate, thiamine, and thiazole pyrophosphate) upon the reconstitution procedure was evaluated. The complete association of sub-units to form active oligomer was attained only when thiamine pyrophosphate was present. It is concluded that both the pyrimidine ring and the pyrophosphate group are required for productive co-enzyme binding and it is proposed that this interaction effects a conformational change which promotes protomer aggregation to form the enzymatically active holoenzyme. In addition data are presented which indicate that the monomer unit is 60 000 +/- 3000 daltons and that the N-terminal amino acid is histidine. Since the molecular weight of the active oligomer is 230 000 it is proposed that pyruvate decarboxylase is a tetramer comprised of four identical or nearly identical monomer units.

Binding Sites↗
PubMed1989

Identification, cloning and characterisation of a new gene required for full pyruvate decarboxylase activity in Saccharomyces cerevisiae.

Biochemical evidence that pyruvate decarboxylase in S. cerevisiae might be constituted from two independently encoded subunits led us to question genetic evidence for a single structural gene. The main evidence for this was that three "structural" mutations appeared to be alleles of the same gene, PDC1 (Schmitt and Zimmermann 1982). We report that one of these mutations (pdc1-30) is not allelic either to other pdc1 alleles or to pdc2 mutations and therefore is has been renamed pdc3-30 thus identifying a new gene, PDC3. We have cloned the PDC3 gene, it represents a unique sequence in the genome and targeted integration shows tight linkage to the PDC3 locus. However, the size, abundance and regulation of the PDC3 transcript suggest that it does not encode a second structural gene. Possible functions for the PDC3 gene product are discussed.

Alleles↗
PubMed1996

Characterization of pyruvate decarboxylase genes from rice.

The pdc1 gene encoding pyruvate decarboxylase has been isolated and sequenced from an IR54 rice genomic library. In contrast to a previously isolated intron-less rice genomic pdc, pRgpdc3, this gene contains five intervening introns in the coding region and corresponds to a cDNA clone, pRcpdc1, isolated from an IR54-cDNA library constructed from anaerobically-induced mRNAs. Comparison of the deduced amino acid sequence of this gene with that of the rice pdc2 and pdc3 showed 88% and 89% similarity, and 78% and 79% identity, respectively. Southern blots indicated that more than three genes constitute the pdc gene family in rice. pdc1 is highly inducible under anaerobic conditions. Rice pdc2 is also inducible by anoxia but to a much lesser extent than pdc1.

Aerobiosis↗
PubMed1993

Effects of metal ions, thiamine diphosphate analogues and subunit interactions on the reconstitution behaviour of pyruvate decarboxylase from brewer's yeast.

The reconstitution of pyruvate decarboxylase starts with reversible binding of thiamine diphosphate and Mg2(+)-ions to the apoenzyme, followed by a rate-limiting conformational change to the catalytically active holoenzyme. Investigations with diphospho-esters of 4-methyl-5-(2-hydroxyethyl)thiazolium derivatives have shown that the diphosphate residue of thiamine diphosphate is the most important part of the coenzyme responsible for the first reversible binding step. Methylation of the N1'-atom of the pyrimidine ring of thiamine diphosphate or 4'-oxythiamine diphosphate prevents the coenzyme from binding stably to the apoenzyme, so that the methylated coenzyme displays no coenzyme activity. In contrast, thiamine diphosphate analogues with bulky residues on the neighbouring C2'-atom of the pyrimidine ring form active holoenzyme complexes. This result shows the essential role of the N1'-atom of thiamine diphosphate in stable cofactor binding. The cofactor binding rate to the dimeric and tetrameric apoenzymes indicates that the cofactor is located in the contact regions of the subunits in the tetrameric enzyme.

Binding Sites↗
PubMed2000

Effects of deletions at the carboxyl terminus of Zymomonas mobilis pyruvate decarboxylase on the kinetic properties and substrate specificity.

The three-dimensional structure of Zymomonas mobilis pyruvate decarboxylase shows that the carboxyl-terminal region of the protein occludes the active site. This observation is consistent with earlier suggestions that the active site is inaccessible to solvent during catalysis. However, the carboxyl-terminal region must move aside to allow entry of the substrate, and again to permit the products to leave. Here we have examined the role of the carboxyl terminus by making 15 variants of the enzyme with serial deletions. The activity is largely unaffected by removal of up to seven residues but deletion of the next two, R561 and S560, results in a drastic loss of activity. Five of these deletion mutants were purified and fully characterized and showed progressive decreases in activity, in the ability to discriminate between pyruvate and larger substrates, and in cofactor affinity. Several substitution mutants at residues R561 and S560 were prepared, purified, and fully characterized. The results indicate important roles for the side-chain of R561 and the backbone atoms of S560. It is suggested that the carboxyl-terminal region of pyruvate decarboxylase is needed to lock in the cofactors and for the proper closure of the active site that is required for discrimination between substrates and for decarboxylation to occur.

Amino Acid Sequence↗