Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen Staphylococcus aureus. In this study we showed through genetic disruption experiments that mvaA, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of S. aureus. Supplementation of media with mevalonate permitted isolation of an auxotrophic mvaA null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The mvaA gene was cloned from S. aureus DNA and expressed with an N-terminal His tag in Escherichia coli. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the S. aureus enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a K i of 320 M, over 10 4 higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.

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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.

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There are two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase: the class I enzymes of eukaryotes and some archaea, and the class II enzymes of certain eubacteria. The activity of the class I Syrian hamster HMG-CoA reductase is regulated by phosphorylation-dephosphorylation of Ser871. Phosphorylation apparently prevents the active site histidine, His865, from protonating the inhibitory coenzyme A thioanion prior to its release from the enzyme. Structural evidence for this hypothesis is, however, lacking. The HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus, whose stability recommends it for physical studies, lacks both a phosphoacceptor serine and a protein kinase recognition motif. Consequently, its activity is not regulated by phosphorylation. We therefore employed site-directed mutagenesis to engineer an appropriately located phosphoacceptor serine and cAMP-dependent protein kinase recognition motif. Substitution of serine for Ala406, the apparent cognate of hamster Ser871, and replacement of Leu403 and Gly404 by arginine created S. solfataricus mutant enzyme L403R/G404R/A406S. The general properties of enzyme L403R/G404R/A406S (K(m) values, V(max), optimal pH and temperature) were essentially those of the wild-type enzyme. Exposure of enzyme L403R/G404R/A406S to [gamma-(32)P]ATP and cAMP-dependent protein kinase was accompanied by incorporation of (32)P(i) and by a parallel decrease in catalytic activity. Subsequent treatment with a protein phosphatase released enzyme-bound (32)P(i) and restored activity to pretreatment levels. The regulatory properties of enzyme L403R/G404R/A406S thus match those of the hamster enzyme. Solution of the three-dimensional structures of the phospho and dephospho forms of this mutant enzyme thus should reveal structural features critical for regulation of the activity of a class I HMG-CoA reductase.