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Journal of Bacteriology, September 2000, p. 5147-5152, Vol. 182, No. 18
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Essentiality, Expression, and Characterization of the Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase of Staphylococcus aureus

E. Imogen Wilding,1 Dong-Yul Kim,2 Alexander P. Bryant,1 Michael N. Gwynn,1,* R. Dwayne Lunsford,1 Damien McDevitt,1 Joseph E. Myers Jr.,1 Martin Rosenberg,1 Daniel Sylvester,1 Cynthia V. Stauffacher,3 and Victor W. Rodwell2

Department of Microbiology, SmithKline Beecham Pharmaceuticals, Collegeville, Pennsylvania 19426,1 and Departments of Biochemistry2 and Biological Sciences,3 Purdue University, West Lafayette, Indiana 47907

Received 21 April 2000/Accepted 12 June 2000

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 Ki of 320 µM, over 104 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.

* Corresponding author. Mailing address: Department of Microbiology, SmithKline Beecham Pharmaceuticals, 1250 South College Rd., Collegeville, PA 19426. Phone: (610) 917-7749. Fax: (610) 917-4989. E-mail: Mick_Gwynn-1{at}sbphrd.com.

Journal of Bacteriology, September 2000, p. 5147-5152, Vol. 182, No. 18
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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