The Role of His113 and His114 in Pyruvate Decarboxylase from <i>Zymomonas Mobilis</i>

Schenk, Gerhard; Leeper, Finian J.; England, Renee; Nixon, P.G.F.; Duggleby, Ronald G.

doi:10.1111/j.1432-1033.1997.t01-1-00063.x

Cited by 48 publications

(69 citation statements)

References 53 publications

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“…The effect of pH on k cat /K m compared to k cat has been well documented for this enzyme by titration curves, with pK a values estimated at 6.23 to 6.45 for the residue involved in modulating substrate binding (21,43). The deprotonation of His113 has been suggested to lead to conformational changes that result in a flexible loop (residues 105 to 112) to close over the active site during catalysis (43). Both His113 and His114 are conserved in all PDC proteins that have been characterized.…”

Section: Resultsmentioning

confidence: 99%

Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene ( pdc ) and Comparison to Bacterial Homologues

Krishnan

Talarico

Ingram

et al. 2002

Appl Environ Microbiol

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Pyruvate decarboxylase (PDC) is the key enzyme in all homo-ethanol fermentations. Although widely distributed among plants, yeasts, and fungi, PDC is absent in animals and rare in bacteria (established for only three organisms). Genes encoding the three known bacterial pdc genes have been previously described and expressed as active recombinant proteins. The pdc gene from Zymomonas mobilis has been used to engineer ethanol-producing biocatalysts for use in industry. In this paper, we describe a new bacterial pdc gene from Zymobacter palmae. The pattern of codon usage for this gene appears quite similar to that for Escherichia coli genes. In E. coli recombinants, the Z. palmae PDC represented approximately 1/3 of the soluble protein.Biochemical and kinetic properties of the Z. palmae enzyme were compared to purified PDCs from three other bacteria. Of the four bacterial PDCs, the Z. palmae enzyme exhibited the highest specific activity (130 U mg of protein ؊1 ) and the lowest K m for pyruvate (0.24 mM). Differences in biochemical properties, thermal stability, and codon usage may offer unique advantages for the development of new biocatalysts for fuel ethanol production.Pyruvate decarboxylase (PDC) is the key enzyme for all homo-fermentative ethanol pathways. This enzyme catalyzes the nonoxidative decarboxylation of pyruvate to acetaldehyde using Mg 2ϩ and thiamine pyrophosphate (TPP) as cofactors. Acetaldehyde is reduced to ethanol by alcohol dehydrogenase (ADH) during NADH oxidation. ADH enzymes are widely distributed throughout nature (41). In contrast, PDC is common to only plants, yeasts, and fungi; it is absent in animals and rare in prokaryotes (25). PDC has been established by cloning and purification for only three bacteria, Zymomonas mobilis (6,7,11,19,33,34), Sarcina ventriculi (28,45), and Acetobacter pasteurianus (40). Though cloned from plants and fungi (25,38), only bacterial PDC enzymes are produced at high levels as active recombinant products (6,10,11,34,40,45). Recombinant Z. mobilis PDC has been used for the metabolic engineering of ethanol pathways in many organisms (4,8,14,16,22,44).An ethanologenic bacterium, Zymobacter palmae, has been reported which appears to contain a PDC enzyme (37). Z. palmae was originally isolated from palm sap (37) and produces ethanol as a primary fermentation product from a variety of hexose sugars and saccharides (20,36). This bacterium is distinct from other ethanol-fermenting bacteria including those belonging to the genera Zymomonas. Zymomonas is well known for production of ethanol from plant sap in tropical areas; however, its fermentable carbohydrates are limited to glucose, fructose, and saccharose (22). In addition to substrate utilization, Zymobacter (55.8 Ϯ 0.4 mol% GϩC) differs dramatically from Zymomonas (48.5 Ϯ 0.5 mol% GϩC) in genome composition. This is consistent with the other phenotypic differences that have been observed, including the type of ubiquinone synthesized and peritrichous versus polar flagellation (37).In this paper, we have establis...

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Section: Resultsmentioning

confidence: 99%

Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene ( pdc ) and Comparison to Bacterial Homologues

Krishnan

Talarico

Ingram

et al. 2002

Appl Environ Microbiol

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“…First and foremost, the active site as such is complete only in the activated state. A vital role has been ascribed to His-113 in ZmPDC, as part of a catalytic dyad supporting aldehyde release (44,50). Its position is equivalent to that of His-114 in ScPDC and KlPDC, respectively, which reorients upon activation.…”

Section: Structural Implicationsmentioning

confidence: 99%

Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

Kutter

Weiss

Wille

et al. 2009

Journal of Biological Chemistry

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The mechanism by which the enzyme pyruvate decarboxylase from two yeast species is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants, or of enzyme complexes from two yeast species, three of them reported here for the first time, provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighboring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, one of which provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies, providing a consistent picture of the structural changes occurring upon enzyme activation.

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“…It is proposed that this hydrogen bond promotes the generation of a resonance form with a positively charged imino-group at the 4 H -position of the pyrimidine ring. The side chain of His481 is close to this charged imino group and it has been suggested that His481 (which corresponds to His113 or His114 in Zymomonas mobilis PDC which have been shown to be involved in various aspects of PDC function [19,139]) may abstract the proton from the imino group, which would increase the pK a of the imino group su ciently that it could in turn remove the proton from the C2 of the thiazolium ring. One problem of this mechanism is that in mammalian TKs His481 is replaced by a glutamine [138], rendering the proposed catalytic function of His481 questionable.…”

Section: Catalytic Mechanism Of Tkmentioning

confidence: 99%

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

Schenk

Duggleby

Nixon

1998

The International Journal of Biochemistry & Cell Biology

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225

164

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This review highlights recent research on the properties and functions of the enzyme transketolase, which requires thiamin diphosphate and a divalent metal ion for its activity. The transketolase-catalysed reaction is part of the pentose phosphate pathway, where transketolase appears to control the non-oxidative branch of this pathway, although the overall¯ux of labelled substrates remains controversial. Yeast transketolase is one of several thiamin diphosphate dependent enzymes whose three-dimensional structures have been determined. Together with mutational analysis these structural data have led to detailed understanding of thiamin diphosphate catalysed reactions. In the homodimer transketolase the two catalytic sites, where dihydroxyethyl groups are transferred from ketose donors to aldose acceptors, are formed at the interface between the two subunits, where the thiazole and pyrimidine rings of thiamin diphosphate are bound. Transketolase is ubiquitous and more than 30 full-length sequences are known. The encoded protein sequences contain two motifs of high homology; one common to all thiamin diphosphate-dependent enzymes and the other a unique transketolase motif. All characterised transketolases have similar kinetic and physical properties, but the mammalian enzymes are more selective in substrate utilisation than the nonmammalian representatives. Since products of the transketolase-catalysed reaction serve as precursors for a number of synthetic compounds this enzyme has been exploited for industrial applications. Putative mutant forms of transketolase, once believed to predispose to disease, have not stood up to scrutiny. However, a modi®cation of transketolase is a marker for Alzheimer's disease, and transketolase activity in erythrocytes is a measure of thiamin nutrition. The cornea contains a particularly high transketolase concentration, consistent with the proposal that pentose phosphate pathway activity has a role in the removal of light-generated radicals. #

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The Role of His113 and His114 in Pyruvate Decarboxylase from Zymomonas Mobilis

Cited by 48 publications

References 53 publications

Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene ( pdc ) and Comparison to Bacterial Homologues

Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene ( pdc ) and Comparison to Bacterial Homologues

Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

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