Enhanced ethanol formation by Clostridium thermocellum via pyruvate decarboxylase.

BACKGROUND: Pyruvate decarboxylase (PDC) is a well-known pathway for ethanol production, but has not been demonstrated for high titer ethanol production at temperatures above 50 °C. RESULT: Here we examined the thermostability of eight PDCs. The purified bacterial enzymes retained 20% of activity after incubation for 30 min at 55 °C. Expression of these PDC genes, except the one from Zymomonas mobilis, improved ethanol production by Clostridium thermocellum. Ethanol production was further improved by expression of the heterologous alcohol dehydrogenase gene adhA from Thermoanaerobacterium saccharolyticum. CONCLUSION: The best PDC enzyme was from Acetobactor pasteurianus. A strain of C. thermocellum expressing the pdc gene from A. pasteurianus and the adhA gene from T. saccharolyticum was able to produce 21.3 g/L ethanol from 60 g/L cellulose, which is 70% of the theoretical maximum yield.

A pyruvate decarboxylase-mediated therapeutic strategy for mimicking yeast metabolism in cancer cells.

Cancer cells have high rates of glycolysis and lactic acid fermentation in order to fuel accelerated rates of cell division (Warburg effect). Here, we present a strategy for merging cancer and yeast metabolism to remove pyruvate, a key intermediate of cancer cell metabolism, and produce the toxic compound acetaldehyde. This approach was achieved by administering the yeast enzyme pyruvate decarboxylase to triple negative breast cancer cells. To overcome the challenges of protein delivery, a nanoparticle-based system consisting of cationic lipids and porous silicon were employed to obtain efficient intracellular uptake. The results demonstrate that the enzyme therapy decreases cancer cell viability through production of acetaldehyde and reduction of lactic acid fermentation.

The bifunctional pyruvate decarboxylase/pyruvate ferredoxin oxidoreductase from Thermococcus guaymasensis.

The hyperthermophilic archaeon Thermococcus guaymasensis produces ethanol as a metabolic end product, and an alcohol dehydrogenase (ADH) catalyzing the reduction of acetaldehyde to ethanol has been purified and characterized. However, the enzyme catalyzing the formation of acetaldehyde has not been identified. In this study an enzyme catalyzing the production of acetaldehyde from pyruvate was purified and characterized from T. guaymasensis under strictly anaerobic conditions. The enzyme had both pyruvate decarboxylase (PDC) and pyruvate ferredoxin oxidoreductase (POR) activities. It was oxygen sensitive, and the optimal temperatures were 85°C and >95°C for the PDC and POR activities, respectively. The purified enzyme had activities of 3.8 ± 0.22 U mg(-1) and 20.2 ± 1.8 U mg(-1), with optimal pH-values of 9.5 and 8.4 for each activity, respectively. Coenzyme A was essential for both activities, although it did not serve as a substrate for the former. Enzyme kinetic parameters were determined separately for each activity. The purified enzyme was a heterotetramer. The sequences of the genes encoding the subunits of the bifunctional PDC/POR were determined. It is predicted that all hyperthermophilic ß -keto acids ferredoxin oxidoreductases are bifunctional, catalyzing the activities of nonoxidative and oxidative decarboxylation of the corresponding ß -keto acids.

Comparison of pyruvate decarboxylases from Saccharomyces cerevisiae and Komagataella pastoris (Pichia pastoris).

Pyruvate decarboxylases (PDCs) are a class of enzymes which carry out the non-oxidative decarboxylation of pyruvate to acetaldehyde. These enzymes are also capable of carboligation reactions and can generate chiral intermediates of substantial pharmaceutical interest. Typically, the decarboxylation and carboligation processes are carried out using whole cell systems. However, fermentative organisms such as Saccharomyces cerevisiae are known to contain several PDC isozymes; the precise suitability and role of each of these isozymes in these processes is not well understood. S. cerevisiae has three catalytic isozymes of pyruvate decarboxylase (ScPDCs). Of these, ScPDC1 has been investigated in detail by various groups with the other two catalytic isozymes, ScPDC5 and ScPDC6 being less well characterized. Pyruvate decarboxylase activity can also be detected in the cell lysates of Komagataella pastoris, a Crabtree-negative yeast, and consequently it is of interest to investigate whether this enzyme has different kinetic properties. This is also the first report of the expression and functional characterization of pyruvate decarboxylase from K. pastoris (PpPDC). This investigation helps in understanding the roles of the three isozymes at different phases of S. cerevisiae fermentation as well as their relevance for ethanol and carboligation reactions. The kinetic and physical properties of the four isozymes were determined using similar conditions of expression and characterization. ScPDC5 has comparable decarboxylation efficiency to that of ScPDC1; however, the former has the highest rate of reaction, and thus can be used for industrial production of ethanol. ScPDC6 has the least decarboxylation efficiency of all three isozymes of S. cerevisiae. PpPDC in comparison to all isozymes of S. cerevisiae is less efficient at decarboxylation. All the enzymes exhibit allostery, indicating that they are substrate activated.

Bifunctionality of the thiamin diphosphate cofactor: assignment of tautomeric/ionization states of the 4'-aminopyrimidine ring when various intermediates occupy the active sites during the catalysis of yeast pyruvate decarboxylase.

Thiamin diphosphate (ThDP) dependent enzymes perform crucial C-C bond forming and breaking reactions in sugar and amino acid metabolism and in biosynthetic pathways via a sequence of ThDP-bound covalent intermediates. A member of this superfamily, yeast pyruvate decarboxylase (YPDC) carries out the nonoxidative decarboxylation of pyruvate and is mechanistically a simpler ThDP enzyme. YPDC variants created by substitution at the active center (D28A, E51X, and E477Q) and on the substrate activation pathway (E91D and C221E) display varying activity, suggesting that they stabilize different covalent intermediates. To test the role of both rings of ThDP in YPDC catalysis (the 4'-aminopyrimidine as acid-base, and thiazolium as electrophilic covalent catalyst), we applied a combination of steady state and time-resolved circular dichroism experiments (assessing the state of ionization and tautomerization of enzyme-bound ThDP-related intermediates), and chemical quench of enzymatic reaction mixtures followed by NMR characterization of the ThDP-bound intermediates released from YPDC (assessing occupancy of active centers by these intermediates and rate-limiting steps). Results suggest the following: (1) Pyruvate and analogs induce active site asymmetry in YPDC and variants. (2) The rare 1',4'-iminopyrimidine ThDP tautomer participates in formation of ThDP-bound intermediates. (3) Propionylphosphinate also binds at the regulatory site and its binding is reflected by catalytic events at the active site 20 Å away. (4) YPDC stabilizes an electrostatic model for the 4'-aminopyrimidinium ionization state, an important contribution of the protein to catalysis. The combination of tools used provides time-resolved details about individual events during ThDP catalysis; the methods are transferable to other ThDP superfamily members.

Reduction of PDC1 expression in S. cerevisiae with xylose isomerase on xylose medium.

Ethanol production using hemicelluloses has recently become a focus of many researchers. In order to promote D: -xylose fermentation, we cloned the bacterial xylA gene encoding for xylose isomerase with 434 amino acid residues from Agrobacterium tumefaciens, and successfully expressed it in Saccharomyces cerevisiae, a non-xylose assimilating yeast. The recombinant strain S. cerevisiae W303-1A/pAGROXI successfully colonized a minimal medium containing D: -xylose as a sole carbon source and was capable of growth in minimal medium containing 2% xylose via aerobic shake cultivation. Although the recombinant strain assimilates D: -xylose, its ethanol productivity is quite low during fermentation with D: -xylose alone. In order to ascertain the key enzyme in ethanol production from D: -xylose, we checked the expression levels of the gene clusters involved in the xylose assimilating pathway. Among the genes classified into four groups by their expression patterns, the mRNA level of pyruvate decarboxylase (PDC1) was reduced dramatically in xylose media. This reduced expression of PDC1, an enzyme which converts pyruvate to acetaldehyde, may cause low ethanol productivity in xylose medium. Thus, the enhancement of PDC1 gene expression may provide us with a useful tool for the fermentation of ethanol from hemicellulose.

The influence of protein concentration on oligomer structure and catalytic function of two pyruvate decarboxylases.

As a general rule protein concentration typical for structural studies differs considerably from that chosen for kinetic investigations. Consequently, structure-function relationships are often postulated without appropriate knowledge, whether the functional behaviour of the enzyme is the same in both protein concentration ranges. To deal with this question, substrate activation kinetics of two well-characterised yeast pyruvate decarboxylases, from Saccharomyces cerevisiae and from Kluyveromyces lactis, were analysed over the broad protein concentration range 2-2,000 microg/mL. Analytical ultracentrifugation and small-angle X-ray scattering were used to analyse the enzymes' oligomer structure in aqueous solution. For the upper part of the concentration range the determined parameters, like catalytic activity, observed substrate activation rates, sedimentation coefficients and scattering parameters are independent on enzyme concentration changes. No indication of protein aggregation is detectable. However, significant changes occur at low enzyme concentration. The catalytically active tetramer dissociates progressively into dimers with comparable catalytic activity, but with significantly accelerated substrate activation.

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.

Pyruvate carboxylase from Mycobacterium smegmatis: stabilization, rapid purification, molecular and biochemical characterization and regulation of the cellular level.

This is the first report on the purification and characterization of an anaplerotic enzyme from a Mycobacterium. The anaplerotic reactions play important roles in the biochemical differentiation of mycobacteria into non-replicating stages. We have purified and characterized a pyruvate carboxylase (PYC) from Mycobacterium smegmatis and cloned and sequenced its gene. We have developed a very rapid and efficient purification protocol that provided PYC with very high specific activities (up to 150 U/mg) that remained essentially unchanged over a month. The enzyme was found to be a homomultimer of 121 kDa subunits, mildly thermophilic, absolutely dependent on acyl-CoAs for activity and inhibited by ADP, by excess Mg(2+), Co(2+), and Mn(2+), by aspartate, but not by glutamate and alpha-ketoglutarate. Supplementation of minimal growth medium with aspartate did not lower the cellular PYC level, rather doubled it; with glutamate the level remained unchanged. These observations would not fit the idea that the M. smegmatis enzyme fulfills a straightforward anaplerotic function; in a closely related organism, Corynebacterium glutamicum, PYC is the major anaplerotic enzyme. Growth on glucose provided 2-fold higher cellular PYC level than that observed with glycerol. The PYCs of M. smegmatis and Mycobacterium tuberculosis were highly homologous to each other. In M. smegmatis, M. tuberculosis and M. lepra, pyc was flanked by a putative methylase and a putative integral membrane protein genes in an identical operon-like arrangement. Thus, M. smegmatis could serve as a model for studying PYC-related physiological aspects of mycobacteria. Also, the ease of purification and the extraordinary stability could make the M. smegmatis enzyme a model for studying the structure-function relationships of PYCs in general. It should be noted that no crystal structure is available for this enzyme of paramount importance in all three domains of life, archaea, bacteria, and eukarya.

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621.

The role of pyruvate metabolism in the triggering of aerobic, alcoholic fermentation in Saccharomyces cerevisiae has been studied. Since Candida utilis does not exhibit a Crabtree effect. this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate carboxylase (EC 6.4.1.1), the pyruvate dehydrogenase complex and pyruvate decarboxylase (EC 4.1.1.1) in cells of glucose-limited chemostat cultures of the two yeasts were compared. In contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to be a cytosolic enzyme in both yeasts. This implies that for anabolic processes, transport of C4-dicarboxylic acids into the mitochondria is required. Isolated mitochondria from both yeasts exhibited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitochondria for pyruvate oxidation and the in situ activity of the pyruvate dehydrogenase complex was similar in both types of mitochondria. The activity of the cytosolic enzyme pyruvate decarboxylase in S. cerevisiae from glucose-limited chemostat cultures was 8-fold that in C. utilis. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeasts was competitively inhibited by inorganic phosphate. The enzyme of S. cerevisiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarboxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 31P-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glycolytic flux and a high level of this enzyme are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.

Molecular and biochemical characterization of bifunctional pyruvate decarboxylases and pyruvate ferredoxin oxidoreductases from Thermotoga maritima and Thermotoga hypogea.

Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea. Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

Purification and characterization of pyruvate decarboxylase from sweet potato roots.

Pyruvate decarboxylase [2-oxo acid carboxy-lyase, EC 4.1.1.1] was isolated from sweet potato roots and was partially purified from healthy and diseased tissues. There was no appreciable difference in properties between the enzymes from healthy and diseased tissues. The molecular weight of the enzyme was found to be 240,000 by polyacrylamide gel electrophoresis. Since sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a molecular weight of 60,000 for the monomeric form of the enzyme, it is likely that sweet potato pyruvate decarboxylase contains 4 single polypeptide chains. The optimal pH of the decarboxylation reaction was 6.1--6.6. The Lineweaver-Burk double reciprocal plot curved upward, and the Hill coefficient was more than 1, with low concentrations of pyruvate. The enzyme was localized in the cytosol fraction. The activity of the enzyme increased in response to black-rot fungus infection, but decreased in response to cutting.

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.

[Fermentation micromethod for the quantitative determination of thiamine diphosphate in biological fluids]. / Fermentativnyi mikrometod kolichestvennogo opredeleniia tiamindifosfata v viologicheskikh zhidkostiakh.

An enzymatic micromethod is proposed for quantification of thiamine biphosphate (TBP) at concentrations from 0.5 ng in 0.1-0.2 ml samples of blood or other biological liquids. The dynamics of TBP degradation in blood was studied depending on the time and conditions of storage. A high efficient complex of alcohol dehydrogenase and apopyruvate decarboxylase was isolated from baker's yeasts that can be successfully used for quantitative detection of TBP. The complex was stabilized for further application to biochemical kits for diagnosis of B1-deficiency.

[The interaction of pyruvate decarboxylase with the quinone-ferricyanide oxidative system]. / Vzaimodeistvie piruvatdekarboksilazy s okislitel'noi sistemoi khinon--ferritsianid.

Inactivation of yeast pyruvate decarboxylase in the presence of substrate and oxidative system containing substituted quinone and ferricyanide has been investigated. It was established that ferricyanide at pH 5.2-6.4 can prevent irreversible inactivation of the pyruvate decarboxylase caused by the concerted action of pyruvate and substituted quinone. The influence of ferricyanide which depends on the redox potential of the substituted quinone is decreasing in a series tetramethyl-p-benzoquinone, trimethyl-p-benzoquinone, 2-methyl-5-isopropyl-p-benzoquinone. It is supposed that the effect of the oxidative system partially converting the nonoxidative to oxidative function of pyruvate decarboxylase is attributed to the oxidation of active acetaldehyde by substituted quinone and reaction of resultant semiquinone radical with ferricyanide.

[Inactivation of yeast pyruvate decarboxylase in the presence of substrate and quinone electron acceptors]. / Inaktivatsiia drozhzhevoi piruvatdekarboksilazy v prisutstvii substrata i khinonovykh aktseptorov élektronov.

The rate constants of paracatalytic inactivation of pyruvate decarboxylase in the presence of 1,4-naphthoquinones and 1,4-benzoquinones are determined by redox potentials of the oxidant. The logarithm of k2 depends hyperbolically on the redox potential of quinone E0(Q/Q-.) with the coefficient of proportionality which approximates 8.4, The absence of considerable deviations in this correlation for the oxidants of different structures with closed values Eo(Q/Q-.) indicates that the enzyme produces no additional steric barrier.

Catalytically active filaments - pyruvate decarboxylase from Neurospora crassa. pH-controlled oligomer structure and catalytic function.

Pyruvate decarboxylase is a key enzyme in organisms whose energy metabolism is based on alcoholic fermentation. The enzyme catalyses the nonoxidative decarboxylation of 2-oxo acids in the presence of the cofactors thiamine diphosphate and magnesium ions. Pyruvate decarboxylase species from yeasts and plant seeds studied to date are allosterically activated by their substrate pyruvate. However, detailed kinetic studies on the enzyme from Neurospora crassa demonstrate for the first time the lack of substrate activation for a yeast pyruvate decarboxylase species. The quaternary structure of this enzyme species is also peculiar because it forms filamentous structures. The complex enzyme structure was analysed using a number of methods, including small-angle X-ray solution scattering, transmission electron microscopy, analytical ultracentrifugation and size-exclusion chromatography. These measurements were complemented by detailed kinetic studies in dependence on the pH.

Enzymatic (R)-phenylacetylcarbinol production in a benzaldehyde emulsion system with Candida utilis cells.

Recent progress in enzymatic (R)-phenylacetylcarbinol (PAC) production has established the need for low cost and efficient biocatalyst preparation. Pyruvate decarboxylase (PDC) added in the form of Candida utilis cells showed higher stability towards benzaldehyde and temperature in comparison with partially purified preparations. In the presence of 50 mM benzaldehyde and at 4 degrees C, a half-life of 228 h was estimated for PDC added as C. utilis cells, in comparison with 24 h for the partially purified preparation. Increasing the temperature from 4 to 21 degrees C for PAC production with C. utilis cells resulted in similar final PAC levels of 39 and 43 g l(-1) (258 and 289 mM), respectively, from initial 300 mM benzaldehyde and 364 mM pyruvate. The overall volumetric productivity was enhanced 2.8-fold, which reflected the 60% shorter reaction time at the higher temperature. Enantiomeric excess values of 98 and 94% for R-PAC were obtained at 4 and 21 degrees C, respectively, and benzyl alcohol (a potential by-product from benzaldehyde) was not formed.

Proteomics analysis of the tombusvirus replicase: Hsp70 molecular chaperone is associated with the replicase and enhances viral RNA replication.

Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown approximately 35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (ssa1 ssa2) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned ssa1 ssa2 mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.