Dual coenzyme specificity of Archaeoglobus fulgidus HMG-CoA reductase

Comparison of the inferred amino acid sequence of orf AF1736 of Archaeoglob us fulgidus to that of Pseudomonas mevalonii HMG-CoA reductase suggested th at AF1736 might encode a Class II HMG-CoA reductase. Following polymerase c hain reaction-based cloning of AF1736 from A. fulgidus genomic DNA and expr ession in Escherichia coli, the encoded enzyme was purified to apparent hom ogeneity and its enzymic properties were determined, Activity was optimal a t 85 degrees C, Delta H-a was 54 kJ/mol, and the statin drug mevinolin inhi bited competitively with HMG-CoA (K, 180 mu M). Protonated forms of His390 and Lys277, the apparent cognates of the active site histidine and lysine o f the P. mevalonii enzyme, appear essential for activity. The mechanism pro posed for catalysis of P, mevalonii HMG-CoA reductase thus appears valid fo r A. fulgidus HMG-CoA reductase. Unlike any other HMG-CoA reductase, the A. fulgidus enzyme exhibits dual coenzyme specificity, pH activity profiles f or all four reactions revealed that optimal activity using NADP(H) occurred at a pH from 1 to 3 units mon acidic than that observed using NAD(H). Kine tic parameters were therefore determined for all substrates for all four ca talyzed reactions using either NAD(H) or NADP(H). NADPH and NADH compete fo r occupancy of a common site. k(cat)[NAD(H)]/k(cat)[NADP(H)] varied from un ity to under 70 for the four reactions, indicative of slight preference for NAD(H). The results indicate the importance of the protonated status of ac tive site residues His390 and Lys277, shown by altered K-M and k(cat) value s, and indicate that NAD(H) and NADP(H) have comparable affinity for the sa me site.