It has been proposed that isoprenoid biosynthesis in several gram-positive cocci depends on the mevalonate pathway for conversion of acetyl coenzyme A to isopentenyl diphosphate. Mevalonate kinase catalyzes a key reaction in this pathway. In this study the enzyme from Staphylococcus aureus was expressed in Escherichia coli, isolated in a highly purified form, and characterized. The overall amino acid sequence of this enzyme was very heterologous compared with the sequences of eukaryotic mevalonate kinases. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration chromatography suggested that the native enzyme is a monomer with a molecular mass of approximately 33 kDa. The specific activity was 12 U/mg, and the pH optimum was 7.0 to 8.5. The apparent K m values for R,S-mevalonate and ATP were 41 and 339 M, respectively. There was substantial substrate inhibition at millimolar levels of mevalonate. The sensitivity to feedback inhibition by farnesyl diphosphate and its sulfur-containing analog, farnesyl thiodiphosphate, was characterized. These compounds were competitive inhibitors with respect to ATP; the K i values were 46 and 45 M for farnesyl diphosphate and its thio analog, respectively. Parallel measurements with heterologous eukaryotic mevalonate kinases indicated that S. aureus mevalonate kinase is much less sensitive to feedback inhibition (K i difference, 3 orders of magnitude) than the human enzyme. In contrast, both enzymes tightly bound trinitrophenyl-ATP, a fluorescent substrate analog, suggesting that there are similarities in structural features that are important for catalytic function.During isoprenoid biosynthesis in most eubacteria the methyl erythritol 4-phosphate pathway is used for production of isopentenyl diphosphate. In contrast, animals, yeast, and archaea produce isopentenyl diphosphate by the better-characterized mevalonate pathway. Recently, it has been reported (23) that the genomes of several gram-positive cocci encode enzymes of the mevalonate pathway and that survival of these bacteria requires this pathway. The genes that have been proposed to encode enzymes of the mevalonate pathway in grampositive cocci are heterologous with the coding sequences for comparable enzymes in higher organisms. However, the function of the mevalonate pathway in these bacteria is supported by characterization of Enterococcus faecalis 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (20), as well as a dual-function protein, acetoacetyl-CoA thiolase/HMG-CoA reductase (9). Recently, phosphomevalonate kinase activity has been observed for a protein from Streptococcus pneumoniae (15). In contrast, there has not been substantial characterization of mevalonate kinase isolated from any grampositive coccus.Mevalonate kinase (EC 2.7.1.36) catalyzes the following reaction (the reaction is divalent cation dependent, and Mn This enzyme is a key enzyme (22) in the mevalonate pathway for biosynthesis of isopentenyl diphosphate. In fact, genetic defects that decrease ...