Structural Determinants of Nucleotide Coenzyme Specificity in the Distinctive Dinucleotide Binding Fold of HMG-CoA Reductase from Pseudomonas mevalonii

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• 作者：Jon A. Friesen ; C. Martin Lawrence ; Cynthia V. Stauffacher ; and Victor W. Rodwell
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：September 17, 1996
• 年：1996
• 卷：35
• 期：37
• 页码：11945 - 11950
• 全文大小：500K
• 年卷期：v.35,no.37(September 17, 1996)
• ISSN：1520-4995

文摘

The 102-residue small domain of the 428-residueNAD(H)-dependent HMG-CoA reductaseof Pseudomonas mevalonii (EC 1.1.1.88) binds NAD(H) ata distinctive, non-Rossmann dinucleotidebinding fold. The three-dimensional structure reveals that Asp146lies close to the 2'-OH of NAD⁺. Toinvestigate the role of this residue in determination of coenzymespecificity, Asp146 was mutated to Ala,Gly, Ser, and Asn. The mutant enzymes were analyzed for theirability to catalyze the oxidative acylationof mevalonate to HMG-CoA using either the natural coenzymeNAD⁺ or the alternate coenzymeNADP⁺.Mutation of Asp146 to Ala or Gly increased the specificity forNADP⁺, expressed as the ratio ofk_cat/K_mfor NADP⁺ tok_cat/K_m forNAD⁺, 1200-fold (enzyme D146G) and 6700-fold (enzymeD146A). Mutationof Asp146 was accompanied by 565-fold (D146G) and 330-fold (D146A)increases in k_cat/K_m forNADP⁺and 2-fold (D146G) and 20-fold (D146A) decreases ink_cat/K_m forNAD⁺. To further improveNADP⁺specificity, Gln147, Leu148, Leu149, or Thr192 of enzyme D146G or D146Awas replaced by lysine orarginine, which could stabilize the 2'-phosphate ofNADP⁺. Enzymes D146G/T192K,D146G/T192R,D146G/L148K, D146A/L148K, and D146A/L148R exhibited 3200-, 4500-,56 000-, 72 000-, and 83 000-fold increases in the specificity for NADP⁺ relative tothe wild-type enzyme.