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-CoA reductase of Pseudomonas mevalonii (EC 1.1.1.88) binds NAD(H) at a distinctive, non-Rossmann dinucleotide binding fold. The three-dimensional structure reveals that Asp146 lies close to the 2'-OH of NAD-. To investigate the role of this residue in determination of coenzyme specificity, Asp146 was mutated to Ala, Gly, Ser, and Asn. The mutant enzymes were analyzed for their ability to catalyze the oxidative acylation 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 kcat/K(m) for NADP+ to kcat/K(m) for NAD+, 1200-fold (enzyme D146G) and 6700-fold (enzyme D146A). Mutation of Asp146 was accompanied by 565-fold (D146G) and 330-fold (D146A) increases in kcat/K(m) for NADP+ and 2-fold (D146G) and 20-fold (D146A) decreases in kcat/K(m) for NAD+. To further improve NADP+ specificity, Gln147, Leu148, Leu149, or Thr192 of enzyme D146G or D146A was replaced by lysine or arginine, which could stabilize the 2'-phosphate of NADP+. Enzymes D146G/T192K, D146G/T192R, D146G/L148K, D146A/L148K, and D146A/L148R exhibited 3200-, 4500-, 56000-, 72000-, and 83000-fold increases in the specificity for NADP+ relative to the wild-type enzyme.
Friesen
J A
JA
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Lawrence
C M
CM
Stauffacher
C V
CV
Rodwell
V W
VW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006224:Cricetinae; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D017354:Point Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011549:Pseudomonas / Q000201:enzymology*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012617:Sea Urchins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D006903:Hydroxymethylglutaryl CoA Reductases