Dual coenzyme specificity of Archaeoglobus fulgidus HMG-CoA reductase.
Comparison of the inferred amino acid sequence of orf AF1736 of Archaeoglobus fulgidus to that of Pseudomonas mevalonii HMG-CoA reductase suggested that AF1736 might encode a Class II HMG-CoA reductase. Following polymerase chain reaction-based cloning of AF1736 from A. fulgidus genomic DNA and expression in Escherichia coli, the encoded enzyme was purified to apparent homogeneity and its enzymic properties were determined. Activity was optimal at 85 degrees C, deltaHa was 54 kJ/mol, and the statin drug mevinolin inhibited competitively with HMG-CoA (Ki 180 microM). Protonated forms of His390 and Lys277, the apparent cognates of the active site histidine and lysine of the P. mevalonii enzyme, appear essential for activity. The mechanism proposed for catalysis of P. mevalonii HMG-CoA reductase thus appears valid for A. fulgidus HMG-CoA reductase. Unlike any other HMG-CoA reductase, the A. fulgidus enzyme exhibits dual coenzyme specificity. pH-activity profiles for all four reactions revealed that optimal activity using NADP(H) occurred at a pH from 1 to 3 units more acidic than that observed using NAD(H). Kinetic parameters were therefore determined for all substrates for all four catalyzed reactions using either NAD(H) or NADP(H). NADPH and NADH compete for occupancy of a common site. k(cat)[NAD(H)]/k(cat)[NADP(H)] varied from unity to under 70 for the four reactions, indicative of slight preference for NAD(H). The results indicate the importance of the protonated status of active site residues His390 and Lys277, shown by altered K(M) and k(cat) values, and indicate that NAD(H) and NADP(H) have comparable affinity for the same site.
Kim
D Y
DY
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Stauffacher
C V
CV
Rodwell
V W
VW
D019609:Archaeoglobus / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008148:Lovastatin / Q000494:pharmacology; D009841:Oligonucleotides; D013379:Substrate Specificity; D013816:Thermodynamics
D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D009841:Oligonucleotides; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases