title: Essentiality, expression, and characterization of the class II 3-hydroxy-3-methylglutaryl coenzyme A reductase of Staphylococcus aureus.
abstract: 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 microM, 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.
authors.affiliation: Department of Microbiology, SmithKline Beecham Pharmaceuticals, Collegeville, Pennsylvania 19426, USA.
authors.forename: E I
authors.initials: EI
authors.lastname: Wilding
authors.forename: D Y
authors.initials: DY
authors.lastname: Kim
authors.forename: A P
authors.initials: AP
authors.lastname: Bryant
authors.forename: M N
authors.initials: MN
authors.lastname: Gwynn
authors.forename: R D
authors.initials: RD
authors.lastname: Lunsford
authors.forename: D
authors.initials: D
authors.lastname: McDevitt
authors.forename: J E
authors.initials: JE
authors.lastname: Myers
authors.forename: M
authors.initials: M
authors.lastname: Rosenberg
authors.forename: D
authors.initials: D
authors.lastname: Sylvester
authors.forename: C V
authors.initials: CV
authors.lastname: Stauffacher
authors.forename: V W
authors.initials: VW
authors.lastname: Rodwell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017353:Gene Deletion; D005798:Genes, Bacterial; D020043:Genes, Essential; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013203:Staphylococcal Infections / Q000382:microbiology; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity
chemical_list: D007527:Isoenzymes; D006903:Hydroxymethylglutaryl CoA Reductases