Essentiality, expression, and characterization of the class II 3-hydroxy-3-methylglutaryl coenzyme A reductase of Staphylococcus aureus

Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gra m-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 fo r in vitro growth of S. aureus. Supplementation of media with mevalonate pe rmitted isolation of an auxotrophic mvaA null mutant that,vas attenuated fo r virulence in a murine hematogenous pyelonephritis infection model. The mv aA. 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 appa rent homogeneity and was shown to be a class II HMG-CoA reductase, the firs t 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 parame ters were determined for all substrates for all four catalyzed reactions us ing 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). p H profiles suggested that His378 and Lys263, the apparent cognates of the a ctive-site histidine and lysine of Pseudomonas mevalonii HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid fo r the S. aureus enzyme. Fluvastatin inhibited competitively with HMG-CoA, w ith a K-i of 320 mu M, over 10(4) higher than that for a class I HMG-CoA re ductase. Bacterial class II HMG-CoA reductases thus are potential targets f or antibacterial agents directed against multidrug-resistant gram-positive cocci.