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Author (up) Huang, Y.; Lu, Z.; Liu, N.; Chen, Y. file  url
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  Title Identification of important residues in diketoreductase from Acinetobacter baylyi by molecular modeling and site-directed mutagenesis Type Journal Article
  Year 2012 Publication Biochimie Abbreviated Journal Biochimie  
  Volume 94 Issue 2 Pages 471-478  
  Keywords 3-Hydroxyacyl CoA Dehydrogenases/chemistry/genetics/metabolism; Acinetobacter/*enzymology/genetics; Amino Acid Motifs; Amino Acid Substitution; Bacterial Proteins/*chemistry/genetics/metabolism; Binding Sites; Biocatalysis; Esters/*metabolism; Humans; Hydrogen-Ion Concentration; Hydroxymethylglutaryl-CoA Reductase Inhibitors/metabolism; Kinetics; Molecular Dynamics Simulation; Molecular Sequence Data; Mutagenesis, Site-Directed; NAD/metabolism; Oxidation-Reduction; Oxidoreductases/*chemistry/genetics/metabolism; Protein Structure, Tertiary; Recombinant Proteins/chemistry/genetics/metabolism; Sequence Homology, Amino Acid; Stereoisomerism  
  Abstract Diketoreductase (DKR) from Acinetobacter baylyi exhibits a unique property of double reduction of a beta, delta-diketo ester with excellent stereoselectivity, which can serve as an efficient biocatalyst for the preparation of an important chiral intermediate for cholesterol lowering statin drugs. Taken the advantage of high homology between DKR and human heart 3-hydroxyacyl-CoA dehydrogenase (HAD), a molecular model was created to compare the tertiary structures of DKR and HAD. In addition to the possible participation of His-143 in the enzyme catalysis by pH profile, three key amino acid residues, Ser-122, His-143 and Glu-155, were identified and mutated to explore the possibility of involving in the catalytic process. The catalytic activities for mutants S122A/C, H143A/K and E155Q were below detectable level, while their binding affinities to the diketo ester substrate and cofactor NADH did not change obviously. The experimental results were further supported by molecular docking, suggesting that Ser-122 and His-143 were essential for the proton transfer to the carbonyl functional groups of the substrate. Moreover, Glu-155 was crucial for maintaining the proper orientation and protonation of the imidazole ring of His-143 for efficient catalysis.  
  Call Number Serial 1415  
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Author (up) Leskovac, V.; Trivic, S.; Anderson, B.M. file  url
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  Title Use of competitive dead-end inhibitors to determine the chemical mechanism of action of yeast alcohol dehydrogenase Type Journal Article
  Year 1998 Publication Molecular and Cellular Biochemistry Abbreviated Journal  
  Volume 178 Issue 1-2 Pages 219-227  
  Keywords yeast; alcohol; dehydrogenase; dead-end inhibitors; mechanism of action; dehydrogenases  
  Abstract In this work, we have postulated a comprehensive and unified chemical mechanism of action for yeast alcohol dehydrogenase (EC 1.1.1.1, constitutive, cytoplasmic), isolated from Saccharomyces cerevisiae. The chemical mechanism of yeast enzyme is based on the integrity of the proton relay system: His-51....NAD+....Thr-48....R.CH2OH(H2>O)....Zn<math>++, stretching from His-51 on the surface of enzyme to the active site zinc atom in the substrate-binding site of enzyme. Further, it is based on extensive studies of steady-state kinetic properties of enzyme which were published recently. In this study, we have reported the pH-dependence of dissociation constants for several competitive dead-end inhibitors of yeast enzyme from their binary complexes with enzyme, or their ternary complexes with enzyme and NAD+ or NADH; inhibitors include: pyrazole, acetamide, sodium azide, 2-fluoroethanol, and 2,2,2-trifluorethanol. The unified mechanism describes the structures of four dissociation forms of apoenzyme, two forms of the binary complex E.NAD+, three forms of the ternary complex E.NAD+.alcohol, two forms of the ternary complex E.NADH.aldehyde and three binary complexes E.NADH. Appropriate pKa values have been ascribed to protonation forms of most of the above mentioned complexes of yeast enzyme with coenzymes and substrates.  
  Call Number Serial 1414  
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