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BMC Structural Biology Nov 2014Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued...
BACKGROUND
Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC), G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance.
RESULTS
This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (K M 0.06 mM at pH 5), high catalytic efficiencies (1.3 • 10(6) M(-1) • s(-1) at pH 5), pHopt of 5.5 and Topt at 45°C. The enzyme is not thermostable (T½ of 18 minutes at 60°C) and the calculated number of bonds between monomers and dimers do not give clear indications for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z. mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 Å for Cα when comparing GdPDC to that of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci, involving the regions Thr341 to Thr352 and Asn499 to Asp503.
CONCLUSIONS
This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in G. diazotrophicus.
Topics: Amino Acids; Bacterial Proteins; Crystallography, X-Ray; Gluconacetobacter; Models, Molecular; Phylogeny; Protein Conformation; Protein Structure, Quaternary; Protein Structure, Tertiary; Pyruvate Decarboxylase; Sarcina; Sequence Homology, Amino Acid; Substrate Specificity; Zymomonas
PubMed: 25369873
DOI: 10.1186/s12900-014-0021-1 -
Biochimica Et Biophysica Acta Nov 2012Thiamin pyrophosphate (TPP) is essential in carbohydrate metabolism in all forms of life. TPP-dependent decarboxylation reactions of 2-oxo-acid substrates result in... (Review)
Review
Thiamin pyrophosphate (TPP) is essential in carbohydrate metabolism in all forms of life. TPP-dependent decarboxylation reactions of 2-oxo-acid substrates result in enamine adducts between the thiazolium moiety of the coenzyme and decarboxylated substrate. These central enamine intermediates experience different fates from protonation in pyruvate decarboxylase to oxidation by the 2-oxoacid dehydrogenase complexes, the pyruvate oxidases, and 2-oxoacid oxidoreductases. Virtually all of the TPP-dependent enzymes, including pyruvate decarboxylase, can be assayed by 1-electron redox reactions linked to ferricyanide. Oxidation of the enamines is thought to occur via a 2-electron process in the 2-oxoacid dehydrogenase complexes, wherein acyl group transfer is associated with reduction of the disulfide of the lipoamide moiety. However, discrete 1-electron steps occur in the oxidoreductases, where one or more [4Fe-4S] clusters mediate the electron transfer reactions to external electron acceptors. These radical intermediates can be detected in the absence of the acyl-group acceptor, coenzyme A (CoASH). The π-electron system of the thiazolium ring stabilizes the radical. The extensively delocalized character of the radical is evidenced by quantitative analysis of nuclear hyperfine splitting tensors as detected by electron paramagnetic resonance (EPR) spectroscopy and by electronic structure calculations. The second electron transfer step is markedly accelerated by the presence of CoASH. While details of the second electron transfer step and its facilitation by CoASH remain elusive, expected redox properties of potential intermediates limit possible scenarios. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.
Topics: Bacterial Proteins; Biocatalysis; Coenzyme A; Electrons; Free Radicals; Iron-Sulfur Proteins; Models, Molecular; Oxidation-Reduction; Pyruvate Synthase; Thiamine Pyrophosphate
PubMed: 22178227
DOI: 10.1016/j.bbapap.2011.11.010 -
The Journal of Biological Chemistry Dec 2022Pyruvate has two major fates upon entry into mitochondria, the oxidative decarboxylation to acetyl-CoA via the pyruvate decarboxylase complex or the biotin-dependent...
Pyruvate has two major fates upon entry into mitochondria, the oxidative decarboxylation to acetyl-CoA via the pyruvate decarboxylase complex or the biotin-dependent carboxylation to oxaloacetate via pyruvate carboxylase (Pcx). Here, we have generated mice with a liver-specific KO of pyruvate carboxylase (Pcx) to understand the role of Pcx in hepatic mitochondrial metabolism under disparate physiological states. Pcx mice exhibited a deficit in hepatic gluconeogenesis and enhanced ketogenesis as expected but were able to maintain systemic euglycemia following a 24 h fast. Feeding a high-fat diet to Pcx mice resulted in animals that were resistant to glucose intolerance without affecting body weight. However, we found that Pcx mice fed a ketogenic diet for 1 week became severely hypoglycemic, demonstrating a requirement for hepatic Pcx for long-term glycemia under carbohydrate-limited diets. Additionally, we determined that loss of Pcx was associated with an induction in the abundance of lysine-acetylated proteins in Pcx mice regardless of physiologic state. Furthermore, liver acetyl-proteomics revealed a biased induction in mitochondrial lysine-acetylated proteins. These data show that Pcx is important for maintaining the proper balance of pyruvate metabolism between oxidative and anaplerotic pathways.
Topics: Animals; Mice; Diet, Ketogenic; Fasting; Gluconeogenesis; Liver; Lysine; Pyruvate Carboxylase; Pyruvic Acid
PubMed: 36441025
DOI: 10.1016/j.jbc.2022.102648 -
Biochemistry and Biophysics Reports Sep 2016Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium has been purified and...
Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium has been purified and characterized ( ~100 s). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85 °C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms.
PubMed: 28955930
DOI: 10.1016/j.bbrep.2016.07.008 -
Acta Crystallographica. Section F,... Sep 2016Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg(2+) ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to...
Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg(2+) ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Å resolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Å and Rr.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were Rwork = 0.186 (0.271 in the highest resolution bin) and Rfree = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions.
Topics: Amino Acid Sequence; Bacterial Proteins; Catalytic Domain; Cations, Divalent; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli; Ethylene Glycol; Gene Expression; Halomonadaceae; Kinetics; Magnesium; Models, Molecular; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Pyruvate Decarboxylase; Pyruvic Acid; Recombinant Proteins; Substrate Specificity; Thiamine Pyrophosphate
PubMed: 27599861
DOI: 10.1107/S2053230X16012012 -
The Plant Journal : For Cell and... Apr 2019Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO and are well known to play a key role in energy supply via...
Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO and are well known to play a key role in energy supply via fermentative metabolism in oxygen-limiting conditions. In addition to their role in fermentation, plant PDCs have also been hypothesized to be involved in aroma formation although, to date, there is no direct biochemical evidence for this function. We investigated the role of PDCs in fruit volatile biosynthesis, and identified a melon pyruvate decarboxylase, PDC1, that is highly expressed in ripe fruits. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could not only decarboxylate pyruvate, but that it also had significant activity toward other straight- and branched-chain α-ketoacids, greatly expanding the range of substrates previously known to be accepted by the plant enzyme. RNAi-mediated transient and stable silencing of PDC1 expression in melon showed that this gene is involved in acetaldehyde, propanal and pentanal production, while it does not contribute to branched-chain amino acid (BCAA)-derived aldehyde biosynthesis in melon fruit. Importantly, our results not only demonstrate additional functions for the PDC enzyme, but also challenge the long standing hypothesis that PDC is involved in BCAA-derived aldehyde formation in fruit.
Topics: Acetaldehyde; Aldehydes; Carboxy-Lyases; Cucumis melo; Fruit; Gene Expression Profiling; Gene Expression Regulation, Plant; Plant Proteins; Pyruvic Acid
PubMed: 30556202
DOI: 10.1111/tpj.14204 -
Plants (Basel, Switzerland) Aug 2023Kiwifruit ( spp.) is susceptible to waterlogging stress. Although abundant wild germplasm resources exist among plants for improving the waterlogging tolerance of...
Kiwifruit ( spp.) is susceptible to waterlogging stress. Although abundant wild germplasm resources exist among plants for improving the waterlogging tolerance of kiwifruit cultivars, the underlying mechanisms remain largely unknown. Here, a comparative study was undertaken using one wild germplasm, Maorenshen ( Dunn, MRS), and one cultivar, Miliang-1 ( var. (A.Chev.) A.Chev. cv. Miliang-1, ML). Under stress, the ML plantlets were seriously damaged with wilted chlorotic leaves and blackened rotten roots, whereas the symptoms of injury in the MRS plantlets were much fewer, along with higher photosynthetic rates, chlorophyll fluorescence characteristics and root activity under stress conditions. However, neither aerenchyma in the root nor adventitious roots appeared in both germplasms upon stress exposure. The activities of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH), as well as their transcript levels, were constitutively higher in MRS than those in ML under both normal and stress conditions. Waterlogging stress significantly enhanced the PDC and ADH enzyme activities in both germplasms, which were 60.8% and 22.4% higher in the MRS roots than those in the ML roots under waterlogging stress, respectively. Moreover, MRS displayed higher activities of antioxidant enzymes, including SOD, CAT, and APX, as well as DPPH-radical scavenging ability, and decreased HO and MDA accumulation under both normal and stress conditions. Our findings suggest that the waterlogging tolerance of the wild germplasm was associated with high PDC and ADH, as well as antioxidant ability.
PubMed: 37571025
DOI: 10.3390/plants12152872 -
Computational and Structural... 2023Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs...
Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs produced from dietary fiber by gut microbiota fermentation and have beneficial effects on human health. Most previous studies on the antitumor mechanisms of SCFAs have focused on specific metabolites or genes involved in antitumor pathways, such as reactive oxygen species (ROS) biosynthesis. In this study, we performed a systematic and unbiased analysis of the effects of acetate, propionate, and butyrate on ROS levels and metabolic and transcriptomic signatures at physiological concentrations in human colorectal adenocarcinoma cells. We observed significantly elevated levels of ROS in the treated cells. Furthermore, significantly regulated signatures were involved in overlapping pathways at metabolic and transcriptomic levels, including ROS response and metabolism, fatty acid transport and metabolism, glucose response and metabolism, mitochondrial transport and respiratory chain complex, one-carbon metabolism, amino acid transport and metabolism, and glutaminolysis, which are directly or indirectly linked to ROS production. Additionally, metabolic and transcriptomic regulation occurred in a SCFAs types-dependent manner, with an increasing degree from acetate to propionate and then to butyrate. This study provides a comprehensive analysis of how SCFAs induce ROS production and modulate metabolic and transcriptomic levels in colon cancer cells, which is vital for understanding the mechanisms of the effects of SCFAs on antitumor activity in colon cancer.
PubMed: 36874158
DOI: 10.1016/j.csbj.2023.02.022 -
Journal of Biochemistry Oct 2004In 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...
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.
Topics: Allosteric Site; Amino Acid Sequence; Base Sequence; Biochemistry; Blotting, Western; Candida glabrata; Catalysis; Chromatography, Gel; Cloning, Molecular; Conserved Sequence; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Glyoxylates; Hot Temperature; Hydrogen-Ion Concentration; Ketones; Kinetics; Models, Chemical; Models, Genetic; Molecular Sequence Data; Plasmids; Protein Isoforms; Pyruvate Decarboxylase; Recombinant Proteins; Recombination, Genetic; Saccharomyces cerevisiae; Sepharose; Temperature; Time Factors
PubMed: 15625313
DOI: 10.1093/jb/mvh141 -
Bioengineered 2015Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae,...
Mannitol is contained in brown macroalgae up to 33% (w/w, dry weight), and thus is a promising carbon source for white biotechnology. However, Saccharomyces cerevisiae, a key cell factory, is generally regarded to be unable to assimilate mannitol for growth. We have recently succeeded in producing S. cerevisiae that can assimilate mannitol through spontaneous mutations of Tup1-Cyc8, each of which constitutes a general corepressor complex. In this study, we demonstrate production of pyruvate from mannitol using this mannitol-assimilating S. cerevisiae through deletions of all 3 pyruvate decarboxylase genes. The resultant mannitol-assimilating pyruvate decarboxylase-negative strain produced 0.86 g/L pyruvate without use of acetate after cultivation for 4 days, with an overall yield of 0.77 g of pyruvate per g of mannitol (the theoretical yield was 79%). Although acetate was not needed for growth of this strain in mannitol-containing medium, addition of acetate had a significant beneficial effect on production of pyruvate. This is the first report of production of a valuable compound (other than ethanol) from mannitol using S. cerevisiae, and is an initial platform from which the productivity of pyruvate from mannitol can be improved.
Topics: Acetic Acid; Gene Deletion; Genes, Fungal; Kinetics; Mannitol; Metabolic Engineering; Pyruvate Decarboxylase; Pyruvic Acid; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 26588105
DOI: 10.1080/21655979.2015.1112472