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Results for “Pyruvate Decarboxylase”

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PubMed1993

Role of cysteines in the activation and inactivation of brewers' yeast pyruvate decarboxylase investigated with a PDC1-PDC6 fusion protein.

Possible roles of the Cys side chains in the activation and inactivation mechanisms of brewers' yeast pyruvate decarboxylase were investigated by comparing the behavior of the tetrameric enzyme pdc1 containing four cysteines/subunit (positions 69, 152, 221, and 222) with that of a fusion enzyme (pdc1-6, a result of spontaneous gene fusion between PDC1 and PDC6 genes) that is 84% identical in sequence with pdc1 and has only Cys221 (the other three Cys being replaced by aliphatic side chains). The two forms of the enzyme are rather similar so far as steady-state kinetic parameters and substrate activation are considered, as tested for activation by the substrate surrogate pyruvamide. Therefore, if a cysteine is responsible for substrate activation, it must be Cys221. The inactivation of the two enzymes was tested with several inhibitors. Methylmethanethiol sulfonate, a broad spectrum sulfhydryl reagent, could substantially inactivate both enzymes, but was slightly less effective toward the fusion enzyme. (p-Nitrobenzoyl)formic acid is an excellent alternate substrate, whose decarboxylation product p-nitrobenzaldehyde inhibited both enzymes possibly at a Cys221, the only one still present in the fusion enzyme. Exposure of the fusion enzyme, just as of pdc1, to (E)-2-oxo-4-phenyl-3-butenoic acid type inhibitors/alternate substrates enabled detection of the enzyme-bound enamine intermediate at 440 nm. However, unlike pdc1, the fusion enzyme was not irreversibly inactivated by these substrates. These substrates are now known to cause inactivation of pdc1 with concomitant modification of one Cys of the four [Zeng, X.; Chung, A.; Haran, M.; Jordan, F. (1991) J. Am. Chem. Soc. 113, 5842-49].(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, Molecular↗

PubMed1993

Multiple pyruvate decarboxylase genes in maize are induced by hypoxia.

Two cDNA clones corresponding to anaerobically induced maize mRNAs were found to have homology to a previously identified maize pyruvate decarboxylase gene. DNA sequencing and RFLP mapping indicate that these cDNAs represent two additional maize pdc genes. Each of the clones is approximately 85% identical in predicted amino acid sequence to the other two. All three clones are induced by hypoxic stress, but with different levels and kinetics of induction.

Amino Acid Sequence↗

PubMed2000

Fermentative metabolism in grape berries: isolation and characterization of pyruvate decarboxylase cDNA and analysis of its expression throughout berry development.

The involvement of pyruvate decarboxylase (PDC) in the control of alcohol production during ripening of fruit tissues under aerobic conditions has been only partially studied. Enzymological studies showed a significant increase in PDC activity during the ripening of oranges and pears, concurrently with the induction of ethanol production. In tomato, on the other hand, the induction of ethanol production and ADH gene expression after the onset of ripening was not accompanied by induction of PDC activity. The isolation of PDC cDNA from fruits has not yet been reported, nor has its expression pattern during fruit development. We report here the isolation of a cDNA clone encoding for a grape PDC and the characterization of its expression throughout berry development. The pattern of PDC gene expression throughout berry development, combined with earlier findings on constitutive PDC activity in the berry, may suggest that PDC is not the limiting factor for the production of ethanol in the berry, which is induced only after the onset of berry ripening. Alternatively, the induction of ADH gene expression, which occurs only after the onset of ripening in both tomatoes and grape berries, may serve as a regulator of ethanol production in response to a ripening-related cue.

Journal Article↗

PubMed1993

The isolation and nucleotide sequence of the pyruvate decarboxylase gene from Kluyveromyces marxianus.

We have determined the nucleotide sequence of a 2360-basepair (bp) region of the Kluyveromyces marxianus genome containing the structural gene for the enzyme pyruvate decarboxylase (PDC). Comparison of the deduced amino-acid sequence of this gene to that of the Saccharomyces cerevisiae PDC genes reveals extensive homology including a motif common to thiamin diphosphate-dependent enzymes.

Amino Acid Sequence↗

PubMed1976

Citrate treatment in a patient with pyruvate decarboxylase deficiency.

A 5-year old boy with intermittent sopor, severe vomiting and ataxia since 2 1/2 years old was described. During attack period the patient showed elevation of blood pyruvate and alanine and also cerebrospinal-fluid pyruvate, whereas during the time free from attack only blood pyruvate was increased. Deficiency of pyruvate decarboxylase was found in patient's leukocyte and in cultured skin fibroblasts. Addition of thiamine in assay medium did not correct the enzyme activity. Oral administration of citrate seemed to be most effective and during the treatment only a mild attack was once observed.

Carboxy-Lyases↗

PubMed1997

Role of Glu51 for cofactor binding and catalytic activity in pyruvate decarboxylase from yeast studied by site-directed mutagenesis.

We investigated the importance of the interaction between the Nl'-atom of the cofactor thiamine diphosphate and glutamic acid residue 51 in pyruvate decarboxylase (EC 4.1. 1.1). The yeast wild type gene PDCl and the respective mutant genes (E51Q and E51A) were expressed in Escherichia coli. The three enzymes were purified to homogeneity. They comigrated as a single band during silver-stained SDS/PAGE with a molecular mass of 60 000 Da. A molecular mass of 61 200 +/- 200 Da was determined by mass spectrometry for the subunit. The native enzyme is a homotetramer as demonstrated by gel filtration experiments. Near- and far-UV CD spectra showed no significant differences for the apoenzyme of the wild type and the mutants. Slight differences in the rate of thiamine diphosphate binding to the apoprotein component were observed between the wild type and the E51Q PDC by CD spectroscopy. Compared to the wild type enzyme, thiamine diphosphate binding at the E51A mutant apoprotein is very slow. Only 0.04% of the catalytic activity of the wild type enzyme was observed for the E51Q mutant; the E51A mutant has no detectable catalytic activity. The S0.5 value for the substrate pyruvate is increased 33-fold for the E51Q mutant. Substrate activation was observed for both the wild type and the E51Q mutant. The interaction between the N1'-atom of the coenzyme and glutamic acid 51 strongly influences the catalytic activity but only moderately the binding of the cofactor to the apoenzyme and the substrate activation rate.

Binding Sites↗

PubMed2001

Continuous production of (R)-phenylacetylcarbinol in an enzyme-membrane reactor using a potent mutant of pyruvate decarboxylase from Zymomonas mobilis.

The optimization of a continuous enzymatic reaction yielding (R)-phenylacetylcarbinol (PAC), an intermediate of the L-ephedrine synthesis, is presented. We compare the suitability of three pyruvate decarboxylases (PDC), PDC from Saccharomyces cerevisiae, PDC from Zymomonas mobilis, and a potent mutant of the latter, PDCW392M, with respect to their application in the biotransformation using acetaldehyde and benzaldehyde as substrates. Among these, the mutant enzyme was the most active and most stable one. The reaction conditions of the carboligation reaction were investigated by determining initial rate velocities with varying substrate concentrations of both aldehydes. From the resulting data a kinetic model was inferred which fits the experimental data with sufficient reliability to deduce the optimal concentrations of both substrates for the enzymatic process. The results demonstrate that the carboligation is most efficiently performed using a continuous reaction system and feeding both aldehydes in equimolar concentration. Initial studies using a continuously operated enzyme-membrane reactor gave (R)-PAC with a space-time yield of 81 g L(-1). d(-1) using a substrate concentration of 50 mM of both aldehydes. The yield was easily increased by cascadation of enzyme-membrane reactors. The new strategy allows the synthesis of (R)-PAC from cheap substrates in an aqueous reaction system. It thereby overcomes the limitation of by-product formation that severely limits the current fermentative process.

Acetaldehyde↗

PubMed2005

Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii.

The unusual physiological properties of archaea (e.g., growth in extreme salt concentration, temperature and pH) make them ideal platforms for metabolic engineering. Towards the ultimate goal of modifying an archaeon to produce bioethanol or other useful products, the pyruvate decarboxylase gene of Zymomonas mobilis (Zm pdc) was expressed in Haloferax volcanii. This gene has been used successfully to channel pyruvate to ethanol in various Gram-negative bacteria, including Escherichia coli. Although the ionic strength of the H. volcanii cytosol differs over 15-fold from that of E. coli, gel filtration and circular dichroism revealed no difference in secondary structure between the ZmPDC protein isolated from either of these hosts. Like the E. coli purified enzyme, ZmPDC from H. volcanii catalyzed the nonoxidative decarboxylation of pyruvate. A decrease in the amount of soluble ZmPDC protein was detected as H. volcanii transitioned from log phase to late stationary phase that was inversely proportional to the amount of pdc-specific mRNA. Based on these results, proteins from non-halophilic organisms can be actively synthesized in haloarchaea; however, post-transcriptional mechanisms present in stationary phase appear to limit the amount of recombinant protein expressed.

Biotechnology↗

PubMed2002

High-throughput assay of ( R)-phenylacetylcarbinol synthesized by pyruvate decarboxylase.

A novel assay has been developed for the detection of ( R)-phenylacetylcarbinol, ( R)-PAC, a chiral intermediate in the industrial synthesis of ephedrine. It is the product of a biotransformation of benzaldehyde catalysed by the enzyme pyruvate decarboxylase. The assay, using 2,3,5-triphenyltetrazolium chloride, enables high-throughput photometric analysis of the activity of the enzyme thus avoiding time-consuming chromatographic procedures.

Acetone↗

PubMed1983

Active site directed irreversible inactivation of brewers' yeast pyruvate decarboxylase by the conjugated substrate analogue (E)-4-(4-chlorophenyl)-2-oxo-3-butenoic acid: development of a suicide substrate.

(E)-4-(4-Chlorophenyl)-2-oxo-3-butenoic acid (CPB) was found to irreversibly inactivate brewers' yeast pyruvate decarboxylase (PDC, EC 4.1.1.1) in a biphasic, sigmoidal manner, as is found for the kinetic behavior of substrate. An expression was derived for two-site irreversible inhibition of allosteric enzymes, and the kinetic behavior of CPB fit the expression for two-site binding. The calculated Ki's of 0.7 mM and 0.3 mM for CPB were assigned to the catalytic site and the regulatory site, respectively. The presence of pyruvic acid at high concentrations protected PDC from inactivation, whereas low concentrations of pyruvic acid accelerated inactivation by CPB. Pyruvamide, a known allosteric activator of PDC, was found to enhance inactivation by CPB. The results can be explained if pyruvamide binds only to a regulatory site, but CPB and pyruvic acid compete for both the regulatory and the catalytic centers. [1-14C]CPB was found to lose 14CO2 concurrently with the inactivation of the enzyme. Therefore, CPB was being turned over by PDC, in addition to inactivating it. CPB can be labeled a suicide-type inactivator for PDC.

Binding Sites↗

PubMed1996

Reduced pyruvate decarboxylase and increased glycerol-3-phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae.

This investigation deals with factors affecting the production of glycerol in Saccharomyces cerevisiae. In particular, the impact of reduced pyruvate-decarboxylase (PDC) and increased NAD-dependent glycerol-3-phosphate dehydrogenase (GPD) levels was studied. The glycerol yield was 4.7 times (a pdc mutant exhibiting 19% of normal PDC activity) and 6.5 times (a strain exhibiting 20-fold increased GPD activity resulting from overexpression of GPD1 gene) that of the wild type. In the strain carrying both enzyme activity alterations, the glycerol yield was 8.1 times higher than that of the wild type. In all cases, the substantial increase in glycerol yield was associated with a reduction in ethanol yield and a higher by-product formation. The rate of glycerol formation in the pdc mutant was, due to a slower rate of glucose catabolism, only twice that of the wild type, and was increased by GPD1 overexpression to three times that of the wild-type level. Overexpression of GPD1 in the wild-type background, however, led to a six- to seven-fold increase in the rate of glycerol formation. The experimental work clearly demonstrates the rate-limiting role of GPD in glycerol formation in S. cerevisiae.

Adenosine Triphosphate↗

PubMed2002

Yeast pyruvate decarboxylase tetramers can dissociate into dimers along two interfaces. Hybrids of low-activity D28A (or D28N) and E477Q variants, with substitution of adjacent active center acidic groups from different subunits, display restored activity.

The tetrameric enzyme yeast pyruvate decarboxylase (YPDC) has been known to dissociate into dimers at elevated pH values. However, the interface along which the dissociation occurs, as well as the fundamental kinetic properties of the resulting dimers, remains unknown. The active sites of YPDC are comprised of amino acid residues from two subunits, a property which we utilize to address the issue as to which dimer interface is cleaved under different conditions of dissociation. Hydroxide-induced dissociation of the active site D28A (or D28N) and E477Q variants, each at least 100 times less reactive than wild-type YPDC, followed by reassociation of D28A (or D28N) and E477Q variants led to a remarkable 35-50-fold increase in activity. This result is possible only if the hydroxide-induced dissociation results in a cleavage along the interface between two subunits so that residues D28 and E477 are now separated. Upon reassociation, one of the two active sites of the hybrid dimer will have both residues substituted, whereas the second one will be of the wild-type phenotype. In contrast to the hydroxide-induced dimers, the urea-induced dissociation recently proposed results in dissociation along dimer-dimer interfaces, without separating the active sites, and therefore, on reassociation, these dimers do not regain activity. The significance of the results is discussed in light of a recently proposed alternating sites mechanism for YPDC. A preparative ion-exchange method is reported for the separation and purification of hybrid enzymes.

Amino Acid Substitution↗

PubMed1993

The 59-kDa polypeptide constituent of 8-10-nm cytoplasmic filaments in Neurospora crassa is a pyruvate decarboxylase.

The fungus Neurospora crassa harbors large amounts of cytoplasmic filaments which are homopolymers of a 59-kDa polypeptide (P59Nc). We have used molecular cloning, sequencing and enzyme activity measurement strategies to demonstrate that these filaments are made of pyruvate decarboxylase (PDC, EC 4.1.1.1), which is the key enzyme in the glycolytic-fermentative pathway of ethanol production in fungi, and in certain plants and bacteria. Immunofluorescence analyses of 8-10-nm filaments, as well as quantitative Northern blot studies of P59Nc mRNA and measurements of PDC activity, showed that the presence and abundance of PDC filaments depends on the metabolic growth conditions of the cells. These findings may be of relevance to the biology of ethanol production by fungi, and may shed light on the nature and variable presence of filament bundles described in fungal cells.

Amino Acid Sequence↗

PubMed1999

Auxins upregulate expression of the indole-3-pyruvate decarboxylase gene in Azospirillum brasilense.

Transcription of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase involved in the biosynthesis of indole-3-acetic acid (IAA), is induced by IAA as determined by ipdC-gusA expression studies and Northern analysis. Besides IAA, exogenously added synthetic auxins such as 1-naphthaleneacetic acid, 2,4-dichlorophenoxypropionic acid, and p-chlorophenoxyacetic acid were also found to upregulate ipdC expression. No upregulation was observed with tryptophan, acetic acid, or propionic acid or with the IAA conjugates IAA ethyl ester and IAA-L-phenylalanine, indicating structural specificity is required for ipdC induction. This is the first report describing the induction of a bacterial gene by auxin.

Azospirillum brasilense↗

PubMed2001

Catalytic acid-base groups in yeast pyruvate decarboxylase. 1. Site-directed mutagenesis and steady-state kinetic studies on the enzyme with the D28A, H114F, H115F, and E477Q substitutions.

The roles of four of the active center groups with potential acid-base properties in the region of pH optimum of pyruvate decarboxylase from Saccharomyces cerevisiae have been studied with the substitutions Asp28Ala, His114Phe, His115Phe, and Glu477Gln, introduced by site-directed mutagenesis methods. The steady-state kinetic constants were determined in the pH range of activity for the enzyme. The substitutions result in large changes in k(cat) and k(cat)/S(0.5) (and related terms), indicating that all four groups have a role in transition state stabilization. Furthermore, these results also imply that all four are involved in some manner in stabilizing the rate-limiting transition state(s) both at low substrate (steps starting with substrate binding and culminating in decarboxylation) and at high substrate concentration (steps beginning with decarboxylation and culminating in product release). With the exception of some modest effects, the shapes of neither the bell-shaped k(cat)/S(0.5)-pH (and related functions) plots nor the k(cat)-pH plots are changed by the substitutions. Yet, the fractional activity still remaining after substitutions virtually rules out any of the four residues as being directly responsible for initiating the catalytic process by ionizing the C2H. There is no effect on the C2 H/D exchange rate exhibited by the D28A and E477Q substitutions. These results strongly imply that the base-induced deprotonation at C2 is carried out by the only remaining base, the iminopyrimidine tautomer of the coenzyme, via intramolecular proton abstraction. The first product is released as CO(2) rather than HCO(3)(-) by both wild-type and E477Q and D28A variants, ruling out several mechanistic alternatives.

Alanine↗

PubMed1985

Pyruvate Decarboxylase from Zea mays L. : I. Purification and Partial Characterization from Mature Kernels and Anaerobically Treated Roots.

Pyruvate decarboxylase (PDC) was purified from mature, dry maize kernels and from roots of anaerobically treated maize seedlings and partially characterized. PDC was purified to a specific activity of 96 units per milligram protein from kernels and to 41 units per milligram protein from root. The subunit molecular masses were estimated to be 61,000 and 60,000 for kernel PDC and 59,000 and 58,000 for root PDC. The pH optimum for each enzyme was 5.8. Since the pH optimum is nearly one pH unit below the value reported for the cytoplasm of anaerobically metabolizing maize roots (pH 6.7 +/- 0.2), we investigated the effects of pH 5.8 and 6.6 on the cooperative kinetics observed for PDC from each source. The maximum Hill coefficients (n(H)) were much greater at each pH for the kernel PDC (pH 5.8, n(H) = 2.5 and pH 6.6, n(H) = 3.2) than for the root PDC (pH 5.8, n(H) = 1.4 and pH 6.6, n(H) = 1.8). The cooperative kinetics observed with respect to pyruvate were asymmetric. Potassium inhibited maize PDC and was competitive with pyruvate (root PDC K(i) = 16 millimolar and kernel PDC K(i) = 10 millimolar).

Journal Article↗

PubMed1987

Alcohol Dehydrogenase and Pyruvate Decarboxylase Activity in Leaves and Roots of Eastern Cottonwood (Populus deltoides Bartr.) and Soybean (Glycine max L.).

Pyruvate decarboxylase (PDC, EC 4.1.1.1) and alcohol dehydrogenase (ADH, EC 1.1.1.1) are responsible for the anaerobic production of acetaldehyde and ethanol in higher plants. In developing soybean embryos, ADH activity increased upon imbibition and then declined exponentially with development, and was undetectable in leaves by 30 days after imbibition. PDC was not detectable in soybean leaves. In contrast, ADH activity remained high in developing cottonwood seedlings, with no decline in activity during development. ADH activity in the first fully expanded leaf of cottonwood was 230 micromoles NADH oxidized per minute per gram dry weight, and increased with leaf age. Maximal PDC activity of cottonwood leaves was 10 micromoles NADH oxidized per minute per gram dry weight. ADH activity in cottonwood roots was induced by anaerobic stress, increasing from 58 to 205 micromoles NADH oxidized per minute per gram dry weight in intact plants in 48 hours, and from 38 to 246 micromoles NADH oxidized per minute per gram dry weight in detached roots in 48 hours. Leaf ADH activity increased by 10 to 20% on exposure to anaerobic conditions. Crude leaf enzyme extracts with high ADH activity reduced little or no NADH when other aldehydes, such as trans-2-hexenal, were provided as substrate. ADH and PDC are constitutive enzyme in cottonwood leaves, but their metabolic role is not known.

Journal Article↗

PubMed2006

The effect of pyruvate decarboxylase gene knockout in Saccharomyces cerevisiae on L-lactic acid production.

A plant- and crop-based renewable plastic, poly-lactic acid (PLA), is receiving attention as a new material for a sustainable society in place of petroleum-based plastics. We constructed a metabolically engineered Saccharomyces cerevisiae that has both pyruvate decarboxylase genes (PDC1 and PDC5) disrupted in the genetic background to express two copies of the bovine L-lactate dehydrogenase (LDH) gene. With this recombinant, the yield of lactate was 82.3 g/liter, up to 81.5% of the glucose being transformed into lactic acid on neutralizing cultivation, although pdc1 pdc5 double disruption led to ineffective decreases in cell growth and fermentation speed. This strain showed lactate productivity improvement as much as 1.5 times higher than the previous strain. This production yield is the highest value for a lactic acid-producing yeast yet reported.

Bioreactors↗