Site‐directed mutagenesis of the ionizable groups in the active site of <i>Zymomonas mobilis</i> pyruvate decarboxylase

Huang, Chang-Yi; Chang, Alan K.; Nixon, P.G.F.; Duggleby, Ronald G.

doi:10.1046/j.1432-1327.2001.02260.x

Cited by 35 publications

(36 citation statements)

References 36 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).…”

Section: Resultsmentioning

confidence: 89%

“…Thus, His113 is a logical candidate for the observed pH-dependent changes in K m observed for all three gram-negative PDCs. However, five crucial residues with ionizable side chains protruding into the active site of Z. mobilis PDC (Asp27, Glu50, His113, His114, and Glu473) were modified to residues that were nonionizable or had altered pK a values (21). The pH dependence of k cat /K m was relatively unaffected by these modifications, suggesting that these residues, including His113, may not be involved.…”

Section: Resultsmentioning

confidence: 99%

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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: 89%

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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“…His-114 and His-115, the next upstream neighbors of loop 104 -113, are part of the active site. For His-114, an essential function in PDC catalysis has been proposed from kinetic studies with accordant variants from yeast and bacteria (7,8,42,43). Tittmann et al (44) postulated a specific role for His-114 (together with Asp-28) during release of the reaction product acetaldehyde.…”

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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“…That said, there is a positional conservation of residues in that His-113 and His-114, the putative equivalents of PpBFDC His-70 and His-281, respectively, occupy similar spatial positions. Mutagenesis of ZmPDC has shown that these two histidine residues and Asp-27, the spatial equivalent of Ser-26, play important roles in catalysis by PDC (6).…”

mentioning

confidence: 99%

Saturation mutagenesis of putative catalytic residues of benzoylformate decarboxylase provides a challenge to the accepted mechanism

Yep

Kenyon

McLeish

2008

Proc. Natl. Acad. Sci. U.S.A.

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Benzoylformate decarboxylase from Pseudomonas putida (PpBFDC) is a thiamin diphosphate-dependent enzyme that carries out the nonoxidative decarboxylation of aromatic 2-keto acids. The x-ray structure of PpBFDC suggested that Ser-26, His-70, and His-281 would play important roles in its catalytic mechanism, and the S26A, H70A, and H281A variants all exhibited greatly impaired catalytic activity. Based on stopped-flow studies with the alanine mutants, it was proposed that the histidine residues acted as acid-base catalysts, whereas Ser-26 was involved in substrate binding and played a significant, albeit less well defined, role in catalysis. While developing a saturation mutagenesis protocol to examine residues involved in PpBFDC substrate specificity, we tested the procedure on His-281. To our surprise, we found that His-281, which is thought to be necessary for protonation of the carbanion/enamine intermediate, could be replaced by phenylalanine with only a 5-fold decrease in k cat. Even more surprising were our subsequent observations (i) that His-70 could be replaced by threonine or leucine with approximately a 30-fold decrease in kcat/Km compared with a 4,000-fold decrease for the H70A variant and (ii) that Ser-26, which forms hydrogen bonds with the substrate carboxylate, could be replaced by threonine, leucine, or methionine without significant loss of activity. These results call into question the assigned roles for Ser-26, His-70, and His-281. Further, they demonstrate the danger in assigning catalytic function based solely on results with alanine mutants and show that saturation mutagenesis is a valuable tool in assessing the role and relative importance of putative catalytic residues. catalytic mechanism ͉ thiamin diphosphate

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Site‐directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase

Cited by 35 publications

References 36 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

Saturation mutagenesis of putative catalytic residues of benzoylformate decarboxylase provides a challenge to the accepted mechanism

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