Dual coenzyme specificity of Archaeoglobus fulgidus HMG‐CoA reductase

Abstract

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 8C, DH a was 54 kJ0mol, and the statin drug mevinolin inhibited competitively with HMG-Coà K i 180 mM!. 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!#0k 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.Keywords: archaeal oxidoreductase; HMG-CoA reductase; mevaldehyde; mevalonate; redox coenzyme specificity; thermostable enzyme Catalysis by HMG-CoA reductase~E.C. 1.1.1.34! of Reaction~1!, the four-electron reductive deacylation of~S!-HMG-CoA to the isoprenoid precursor~R!-mevalonate, proceeds in three stages, the first and third of which are reductive. The putative intermediates mevaldyl-CoA and mevaldehyde remain enzyme-bound during the course of the reaction. HMG-CoA reductase also catalyzes Reactions 2! and~3!, two 2-electron reactions of free mevaldehyde that appear to model the third stage and the reverse of the second and first stages of Reaction~1!, respectively, and Reaction~4!, the four-electron oxidative acylation of~R!-mevalonate to~S!-HMG-CoA, the reverse of Reaction~1!~Bochar et al., 1999a!.~1 ! Reprint requests to: Victor W. Rodwell, Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907; e-mail: vrodwell@purdue.edu. Abbreviations: CoASH, coenzyme A; HMG, 3-hydroxy-3-methylglutaryl; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The suffixes P and A refer to residues from the HMG-CoA reductases of Pseudomonas mevalonii and of Archaeoglobus fulgidus, respectively.

Enterococcus faecalis Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis

“…As anticipated for an enzyme from a mesophile, the temperature optimum was 37°C. The calculated activation energy ⌬H a of 14 kcal is similar to the ⌬H a of 13 kcal for A. fulgidus HMG-CoA reductase (12), suggesting that the same step is rate limiting for both enzymes. All four reactions catalyzed by HMG-CoA reductase exclusively employed NADP(H) as the coenzyme.…”

“…Table 2 compares these data to those for other characterized HMG-CoA reductases. class II enzymes and in the nanomolar range for the class I enzymes (12,26). Fluvastatin inhibited reaction 3 competitive with HMG-CoA with a K i of 0.5 mM (Fig.…”

“…However, the mvaE gene product represents the first bifunctional enzyme of isopentenyl diphosphate bio- The sequence-based inference that E. faecalis HMG-CoA reductase is a class II enzyme was confirmed by the 0.5 mM K i value for inhibition of reaction 3 by the statin drug fluvastatin. This K i value is diagnostic of a class II enzyme (1,12). As for both classes of the enzyme, inhibition was competitive with HMG-CoA.…”

“…Crystal structures of both classes of the enzyme have been solved (10,15). Relative to the class I HMG-CoA reductases, those of class II are over four orders of magnitude less sensitive to inhibition by statin drugs (1,12,26). Enterococci are a major cause of nosocomial infections, and Enterococcus faecalis is responsible for about 85% of all enterococcal infections.…”

Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

“…Mevinolin, along with its analog simvastatin, is a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase, an enzyme essential for archaeal membrane lipid biosynthesis (11,36). HMG-CoA reductases have been extensively examined from a number of archaeal species (9,10,31). An overexpression construct of the HMG-CoA reductase gene can be expected to be applicable as a marker gene.…”