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PubMed1976

Studies on the mechanism and stereochemical properties of the oxalacetate decarboxylase activity of pyruvate kinase.

When cod fish muscle oxalacetate decarboxylase catalyzes the decarboxylation of oxalacetate in the presence of NaBH4, L-lactate results from the reduction of enzyme-bound pyruvate. However, D-lactate results when borohydride reduces the binary enzyme-pyruvate complex formed by adding pyruvate from solution, as reported by others. This observation suggests that there are alternate mechanisms for reduction that are either kinetically or sterically determined for the E-pyruvate forms produced in the two directions. In the process of investigating the mechanism of reduction, the cod fish muscle decarboxylase was discovered to be identical with pyruvate kinase. Decarboxylase activity appears to take place at a site which overlaps the phosphoenolpyruvate binding site on this enzyme, as discussed in the following paper. Crystalline rabbit muscle pyruvate kinase also contains significant decarboxylase activity indicating that the two reactions may be structurally related functions. In the presence of K+, orthophosphate, or ATP the rabbit muscle enzyme catalyzes the detritiation of enzyme-bound pyruvate formed during decarboxylation before release of pyruvate from the enzyme, in analogy with the detritiation of pyruvate formed from P-[3-3/]enolpyruvate in the kinase reaction. This observation is consistent with the formation of an enolpyruvate intermediate common to the kinetic pathways of both reactions. Since the decarboxylase reac.tion is completely stereospecific, within the limits of detection, going with retention of configuration, the protonation of the enolpyruvate intermediate is completely determined by the enzyme as is the case with the enolpyruvate intermediate generated from P-enolpyruvate in the kinase reaction.

Animals↗

PubMed2000

An investigation of the metabolism of isoleucine to active Amyl alcohol in Saccharomyces cerevisiae.

The metabolism of isoleucine to active amyl alcohol (2-methylbutanol) in yeast was examined by the use of (13)C nuclear magnetic resonance spectroscopy, combined gas chromatography-mass spectrometry, and a variety of mutants. From the identified metabolites a number of routes between isoleucine and active amyl alcohol seemed possible. All involved the initial decarboxylation of isoleucine to alpha-keto-beta-methylvalerate. The first, via branched chain alpha-ketoacid dehydrogenase to alpha-methylbutyryl-CoA, was eliminated because abolition of branched-chain alpha-ketoacid dehydrogenase in an lpd1 disruption mutant did not prevent the formation of active amyl alcohol. However, the lpd1 mutant still produced large amounts of alpha-methylbutyrate which initially seemed contradictory because it had been assumed that alpha-methylbutyrate was derived from alpha-methylbutyryl-CoA via acyl-CoA hydrolase. Subsequently it was observed that alpha-methylbutyrate arises from the non-enzymic oxidation of alpha-methylbutyraldehyde (the immediate decarboxylation product of alpha-keto-beta-methylvalerate). Mutant studies showed that one of the decarboxylases encoded by PDC1, PDC5, PDC6, YDL080c, or YDR380w must be present to allow yeast to utilize alpha-keto-beta-methylvalerate. Apparently, any one of this family of decarboxylases is sufficient to allow the catabolism of isoleucine to active amyl alcohol. This is the first demonstration of a role for the gene product of YDR380w, and it also shows that the decarboxylation steps for each alpha-keto acid in the catabolic pathways of leucine, valine, and isoleucine are accomplished in subtly different ways. In leucine catabolism, the enzyme encoded by YDL080c is solely responsible for the decarboxylation of alpha-ketoisocaproate, whereas in valine catabolism any one of the isozymes of pyruvate decarboxylase will decarboxylate alpha-ketoisovalerate.

Amino Acid Sequence↗

PubMed1996

Pyruvate metabolism in Saccharomyces cerevisiae.

In yeasts, pyruvate is located at a major junction of assimilatory and dissimilatory reactions as well as at the branch-point between respiratory dissimilation of sugars and alcoholic fermentation. This review deals with the enzymology, physiological function and regulation of three key reactions occurring at the pyruvate branch-point in the yeast Saccharomyces cerevisiae: (i) the direct oxidative decarboxylation of pyruvate to acetyl-CoA, catalysed by the pyruvate dehydrogenase complex, (ii) decarboxylation of pyruvate to acetaldehyde, catalysed by pyruvate decarboxylase, and (iii) the anaplerotic carboxylation of pyruvate to oxaloacetate, catalysed by pyruvate carboxylase. Special attention is devoted to physiological studies on S. cerevisiae strains in which structural genes encoding these key enzymes have been inactivated by gene disruption.

Acetaldehyde↗

PubMed1978

Intramolecular coupling of active sites in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The intramolecular passage of substrate between the component enzymes of the pyruvate dehydrogenase multienzyme complex of Escherichia coli was examined. A series of partly reassembled complexes, varying only in their E1 (pyruvate decarboxylase, EC 1.2.4.1) content, was incubated with pyruvate in the absence of CoA, conditions under which the lipoic acid residues covalently bound to the E2 (lipoate acetyltransferase, EC2.3.1.12) chains of the complex become reductively acetylated, and the reaction then ceases. The fraction of E2 chains thus acetylated was estimated by specific reaction of the thiol groups in the acetyl-lipoic acid moieties with N-ethyl[2,3-14C]maleimide. The simplest interpretation of the results was that a single E1 dimer is capable of catalysing the rapid acetylation of 8-12 E2 chains, in good agreement with the results of Bates, Danson, Hale, Hooper & Perham [(1977) Nature (London) 268, 313-316]. This novel functional connexion of active sites must be brought about by transacetylation reactions between lipoic acid residues of neighbouring E2 chains in the enzyme complex. There was also a slow transacylation process between the rapidly acetylated lipoic acid residues and those that did not react in the initial, faster phase. This interaction was not investigated in detail, since it is too slow to be of kinetic significance in the normal enzymic reaction.

Binding Sites↗

PubMed2002

Flux through citrate synthase limits the growth of ethanologenic Escherichia coli KO11 during xylose fermentation.

Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD(+). Expression of low-K(m) Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.

Citrate (si)-Synthase↗

PubMed1996

Stabilization of pet operon plasmids and ethanol production in Escherichia coli strains lacking lactate dehydrogenase and pyruvate formate-lyase activities.

In the last decade, a major goal of research in biofuels has been to metabolically engineer microorganisms to ferment multiple sugars from biomass or agricultural wastes to fuel ethanol. Escherichia coli strains genetically engineered to contain the pet operon (Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase B genes) produce high levels of ethanol. Strains carrying the pet operon in plasmid (e.g., E. coli B/pLOI297) or in chromosomal (e.g., E. coli KO11) sites require antibiotics in the media to maintain genetic stability and high ethanol productivity. To overcome this requirement, we used the conditionally lethal E. coli strain FMJ39, which carries mutations for lactate dehydrogenase and pyruvate formate lyase and grows aerobically but is incapable of anaerobic growth unless these mutations are complemented. E. coli FBR1 and FBR2 were created by transforming E. coli FMJ39 with the pet operon plasmids pLOI295 and pLOI297, respectively. Both strains were capable of anaerobic growth and displayed no apparent pet plasmid losses after 60 generations in serially transferred (nine times) anaerobic batch cultures. In contrast, similar aerobic cultures rapidly lost plasmids. In high-cell-density batch fermentations, 3.8% (wt/vol) ethanol (strain FBR1) and 4.4% (wt/vol) ethanol (strain FBR2) were made from 10% glucose. Anaerobic, glucose-limited continuous cultures of strain FBR2 grown for 20 days (51 generations; 23 with tetracycline and then 28 after tetracycline removal) showed no loss of antibiotic resistance. Anaerobic, serially transferred batch cultures and high-density fermentations were inoculated with cells taken at 57 generations from the previous continuous culture. Both cultures continued to produce high levels of ethanol in the absence of tetracycline. The genetic stability conferred by selective pressure for pet-containing cells without requirement for antibiotics suggests potential commercial suitability for E. coli FBR1 and FBR2.

Acetyltransferases↗

PubMed1976

[Study of certain carbohydrate metabolism enzymes in an active oxytetracycline producer strain and in an inactive mutant in relation to antibiotic biosynthesis].

The activity of the glycolysis enzymes, i.e. aldolase and pyruvate decarboxylase and the enzymes of the pentose cycle, i.e. transketolase were investigated in the process of cultivation of an active strain and inactive mutant of Act. rimosus under conditions favourable for oxytetracycline biosynthesis on starch medium and under unfavourable conditions on glucose medium. It was shown that the aldolase and transketolase activity in the inactive mutant was higher on the starch medium as compared to the active strain, while the activity of pyruvate dekarboxylase was lower. The above difference between the both strains was preserved on the glucose medium and the activity of aldolase and transketolase in both strains increased, while the activity of pyruvate dekarboxylase remained at the same level.

Carbohydrate Metabolism↗

PubMed1981

Petite-negative mutants of Saccharomyces cerevisiae.

A series of yeast mutants has been isolated with the inability to grow on fermentable carbon sources whilst growing normally on ethanol media. One of the mutants, namely MC16/206 lacks pyruvate decarboxylase activity and does not grow on glucose at 37 degrees C but grows on both ethanol and glucose at 27 degrees C. In this strain rho- petites are non-viable.

Alcohol Oxidoreductases↗

PubMed2000

Fermentative capacity in high-cell-density fed-batch cultures of baker's yeast.

High-cell-density fed-batch processes for bakers' yeast production will involve a low-average-specific growth rate due to the limited oxygen-transfer capacity of industrial bioreactors. The relationship between specific growth rate and fermentative capacity was investigated in aerobic, sucrose-limited fed-batch cultures of an industrial bakers' yeast strain. Using a defined mineral medium, biomass concentrations of 130 g dry weight/L were reproducibly attained. After an initial exponential-feed phase (mu = 0.18 h(-1)), oxygen-transfer limitation necessitated a gradual decrease of the specific growth rate to ca. 0.01 h(-1). Throughout fed-batch cultivation, sugar metabolism was fully respiratory, with a biomass yield of 0.5 g biomass/g sucrose(-1). Fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions with excess glucose) showed a strong positive correlation with specific growth rate. The fermentative capacity observed at the end of the process (mu = 0.01 h(-1)) was only half that observed during the exponential-feed phase (mu = 0.18 h(-1)). During fed-batch cultivation, activities of glycolytic enzymes, pyruvate decarboxylase and alcohol dehydrogenase in cell extracts did not exhibit marked changes. This suggests that changes of fermentative capacity during fed-batch cultivation were not primarily caused by regulation of the synthesis of glycolytic enzymes.

Alcohol Dehydrogenase↗

PubMed2005

High-level acetaldehyde production in Lactococcus lactis by metabolic engineering.

Efficient conversion of glucose to acetaldehyde is achieved by nisin-controlled overexpression of Zymomonas mobilis pyruvate decarboxylase (pdc) and Lactococcus lactis NADH oxidase (nox) in L. lactis. In resting cells, almost 50% of the glucose consumed could be redirected towards acetaldehyde by combined overexpression of pdc and nox under anaerobic conditions.

Acetaldehyde↗

PubMed2005

Multiple modes of active center communication in thiamin diphosphate-dependent enzymes.

Detection of interaction between cofactors at the active centers of homodimeric and homotetrameric enzymes is usually elusive by steady-state kinetic approaches and requires protein variants where such interactions are diminished or exaggerated. In this Account, evidence for active-center interactions will be presented for the following thiamin diphosphate-dependent enzymes: yeast pyruvate decarboxylase, benzoylformate decarboxylase, and examples from the 2-oxoacid dehydrogenase multienzyme complex class. The dissymmetry of active sites is especially evident in the X-ray structures of these enzymes with substrate/substrate analogues bound. Perturbations that reveal active center communication include use of chromophoric substrates and substitutions of active center residues on putative pathways.

Binding Sites↗

PubMed2000

Quantitation of low molecular mass substrates and products of enzyme catalyzed reactions using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Relative peak-height ratios of products to substrates determined by MALDI-TOFMS allow the quantitative analysis of enzyme catalyzed reactions for screening purposes. Two examples were investigated: the first one was a lipase catalyzed reaction which produces 2-methoxy-N-[(1R)-1-phenylethyl]acetamide (MET) using rac-alpha-phenylethylamine (PEA) as substrate. The second one was the pyruvate decarboxylase catalyzed formation of (1R)-1-hydroxy-1-phenyl-2-propanone (PAC) with benzaldehyde (BzA) as substrate. Here the corresponding oximes were analyzed after derivatization using hydroxylamine. The standard curves (r2 = 0.985 for MET, r2 = 0.991 for PAC) were linear over two orders of magnitude for MET and PAC concentrations. After optimization of the sample preparation an average relative standard deviation of 12.5% was obtained in both cases.

Acetamides↗

PubMed1989

Linked scans of peptides and protein digests: amino acid sequence determination of components of complex mixtures.

Two-sector linked-scan analysis of an unpurified proteolytic digest of a pyruvate decarboxylase enzyme (60,000 Da) has allowed the discovery and assignment of an amino-terminal post-translational modification and processing event. A difference in amino acid sequence from that predicted by a recently published nucleotide sequence has also been found. These results illustrate both the use and considerable potential of linked-scan methods for the analysis of complex biopolymer mixtures.

Amino Acid Sequence↗

PubMed1995

Transient responses of Candida utilis to oxygen limitation: regulation of the Kluyver effect for maltose.

The facultatively fermentative yeast Candida utilis exhibits the Kluyver effect for maltose: this disaccharide is respired and assimilated but, in contrast to glucose, it cannot be fermented. To study the mechanism of the Kluyver effect, metabolic responses of C. utilis to a transition from aerobic, sugar-limited growth to oxygen-limited conditions were studied in chemostat cultures. Unexpectedly, the initial response of maltose-grown cultures to oxygen limitation was very similar to that of glucose-grown cultures. In both cases, alcoholic fermentation occurred after a lag phase of 1 h, during which glycerol, pyruvate and D-lactate were the main fermentation products. After ca. 10 h the behaviour of the maltose- and glucose-grown cultures diverged: ethanol disappeared from the maltose-grown cultures, whereas fermentation continued in steady-state, oxygen-limited cultures grown on glucose. The disappearance of alcoholic fermentation in oxygen-limited chemostat cultures growing on maltose was not due to a repression of the synthesis of pyruvate decarboxylase and alcohol dehydrogenase. The results demonstrate that the Kluyver effect for maltose in C. utilis does not reflect an intrinsic inability of this yeast to ferment maltose, but is caused by a regulatory phenomenon that affects a key enzyme in maltose metabolism, probably the maltose carrier. The observed kinetics indicate that this regulation occurs at the level of enzyme synthesis rather than via modification of existing enzyme activity.

Alcohol Dehydrogenase↗

PubMed1994

Effects of oxygen limitation on sugar metabolism in yeasts: a continuous-culture study of the Kluyver effect.

Growth and metabolite formation were studied in oxygen-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 growing on glucose or maltose at a dilution rate of 0.1 h-1. With either glucose or maltose S. cerevisiae could be grown under dual limitation of oxygen and sugar. Respiration and alcoholic fermentation occurred simultaneously and the catabolite fluxes through these processes were dependent on the magnitude of the oxygen feed. C. utilis could also be grown under dual limitation of glucose and oxygen. However, at very low oxygen feed rates (i.e. below 4 mmol l-1 h-1) growth was limited by oxygen only, as indicated by the high residual glucose concentration in the culture. In contrast to S. cerevisiae, C. utilis could not be grown anaerobically at a dilution rate of 0.1 h-1. With C. utilis absence of oxygen resulted in wash-out, despite the presence of ergosterol and Tween-80 in the growth medium. The behaviour of C. utilis with respect to maltose utilization in oxygen-limited cultures was remarkable: alcoholic fermentation did not occur and the amount of maltose metabolized was dependent on the oxygen supply. Oxygen-limited cultures of C. utilis growing on maltose always contained high residual sugar concentrations. These observations throw new light on the so-called Kluyver effect. Apparently, maltose is a non-fermentable sugar for C. utilis CBS 621, despite the fact that it can serve as a substrate for growth of this facultatively fermentative yeast. This is not due to the absence of key enzymes of alcoholic fermentation. Pyruvate decarboxylase and alcohol dehydrogenase were present at high levels in maltose-utilizing cells of C. utilis grown under oxygen limitation. It is concluded that the Kluyver effect, in C. utilis growing on maltose, results from a regulatory mechanism that prevents the sugar from being fermented. Oxygen is not a key factor in this phenomenon since under oxygen limitation alcoholic fermentation of maltose was not triggered.

Aerobiosis↗

PubMed2002

The ethanolic fermentation pathway supports respiration and lipid biosynthesis in tobacco pollen.

Rapid pollen tube growth requires a high rate of sugar metabolism to meet energetic and biosynthetic demands. Previous work on pollen sugar metabolism showed that tobacco pollen carry out efficient ethanolic fermentation concomitantly with a high rate of respiration (Bucher et al., 1995). Here we show that the products of fermentation, acetaldehyde and ethanol, are further metabolised in a pathway that bypasses mitochondrial PDH. The enzymes involved in this pathway are pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthetase. Radiolabelling experiments show that during tobacco pollen tube growth label of 14C-ethanol is incorporated into CO2 as well as into lipids and other higher molecular weight compounds. A role for the glyoxylate cycle appears unlikely since activity of malate synthase, a key enzyme of the glyoxylate cycle, could not be detected.

Acetaldehyde↗

PubMed2006

[Effects of 24-epibrassinolide on antioxidant system and anaerobic respiratory enzyme activities in cucumber roots under hypoxia stress].

Seedlings of a hypoxia-resistant cultivar, Lübachun No.4, and a hypoxia-sensitive cultivar, Zhongnong No.8, were applied by feeding roots. The effects of exogenous 24-epibrassinolide (EBR) on lipid peroxidation, activities of major antioxidant enzymes and anaerobic respiratory enzymes and their time courses were investigated in cucumber seedling roots under hypoxia stress. Root growth was significantly inhibited under hypoxia stress, which was improved by exogenous EBR application (Figs.1,2). O(-*)(2) and H(2)O(2) level and lipid peroxidation were significantly increased in the roots upon exposure to hypoxia stress, which were inhibited by exogenous EBR application (Fig.3). EBR treatment increased SOD and POD activities under hypoxia stress (Fig.4). Hypoxia enhanced the activities of lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) whereas EBR treatment significantly increased ADH activity, but decreased LDH activity (Fig.5). There was no significant difference in CAT and PDC activities between the hypoxia stress treatment and "hypoxia+EBR" treatment (Fig.4,5). From the results obtained in this work it can be concluded that oxidative damage on seedling roots by hypoxia stress can be considerably alleviated, and alcohol fermentation can be enhanced by EBR treatment.

Alcohol Dehydrogenase↗

PubMed2003

NMR analysis of covalent intermediates in thiamin diphosphate enzymes.

Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using (1)H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.

Binding Sites↗