STRUCTURAL DETERMINANTS OF NUCLEOTIDE COENZYME SPECIFICITY IN THE DISTINCTIVE DINUCLEOTIDE BINDING FOLD OF HMG-COA REDUCTASE FROM PSEUDOMONAS-MEVALONII

The 102-residue small domain of the 428-residue NAD(H)-dependent HMG-C oA reductase of Pseudomonas mevalonii (EC 1.1.1.88), binds NAD(H) at a distinctive, non-Rossmann dinucleotide binding fold. The three-dimens ional structure reveals that Asp146 lies close to the 2'-OH of NAD(+). To investigate the role of this residue in determination of coenzyme specificity, Asp 146 was mutated to Ala, Gly, Ser, and Asn. The mutant enzymes were analyzed for their ability to catalyze the oxidative acy lation of mevalonate to HMG-CoA using either the natural coenzyme NAD( +) or the alternate coenzyme NADP(+). Mutation of Asp146 to Ala or Gly increased the specificity for NADP(+), expressed as the ratio of k(ca t)/K-m for NADP(+) to k(cat)/K-m for NAD(+), 1200-fold (enzyme D146G) and 6700-fold (enzyme D146A). Mutation of Asp 146 was accompanied by 5 65-fold (D146G) and 330-fold (D146A) increases in k(cat)/K-m for NADP( +) and 2-fold (D146G) and 20-fold (D146A) decreases in k(cat)/K-m for NAD(+). To further improve NADP(+) specificity, Gln147, Leu148, Leu149 , or Thr192 of enzyme D146G or D146A was replaced by lysine or arginin e, which could stabilize the 2'-phosphate of NADP(+). Enzymes D146G/T1 92K, D146G/T192R, D146G/L148K, D146A/L148K, and D146A/L148R exhibited 3200-, 4500-, 56 000-, 72 000-, and 83 000-fold increases in the speci ficity for NADP(+) relative to the wild-type enzyme.