Staphylococcus aureus Mevalonate Kinase: Isolation and Characterization of an Enzyme of the Isoprenoid Biosynthetic Pathway

Abstract

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 ...

Isoprenoid biosynthesis in bacterial pathogens

“…Initial attempts to delete this gene were unsuccessful, and the mevalonate pathway mutant could only be recovered when the medium was supplemented with exogenous mevalonate (Wilding et al, 2000a). Voynova et al (2004) (Laupitz et al, 2004). Takagi et al (2004) identified the ypgA gene product as a type II isomerase, which was found to be a non-essential gene in Bacillus subtilis.…”

Characterization of a Feedback-Resistant Mevalonate Kinase from the Archaeon Methanosarcina mazei

“…Previously the small-molecule regulation of MVKs could be divided into two classes. The first class is inhibited by metabolites downstream of the diphosphomevalonate decarboxylase reaction (IPP, DMAPP, GPP, FPP, and longer chain isoprenoids) (9,16,18,31). The regulation of a eukaryotic MVK isolated from pig liver was first reported by Dorsey and Porter in 1968 (9).…”

“…Two pathways for the biosynthesis of these central metabolites have been described, the mevalonate pathway (28) and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (25). The mevalonate pathway typically is found in animals, plants, and in many Gram-positive bacteria, including Streptococcus pneumoniae (17,31,32). Some enzymes of the mevalonate pathway also have been identified in archaea; however, the complete pathway has not been elucidated (27).…”

“…Organisms manage these tasks through the intricate regulation of isoprenoid-producing pathways (15). MVK was demonstrated to be an important regulatory point in the mevalonate pathway in both bacteria (1,2,31) and eukaryotes (4,9,13,16,18). Previously the small-molecule regulation of MVKs could be divided into two classes.…”

In Vitro and In Vivo Validation of ligA and tarI as Essential Targets in Staphylococcus aureus

“…However, S. aureus-like other gram-positive cocci, such as Streptococcus pneumoniae and Enterococcus faecalis-does not possess the DXP pathway for isoprenoid biosynthesis. Instead, these species use the mevalonate pathway (13,14). Meanwhile, there is increasing evidence that tarI is involved in teichoic acid synthesis in S. aureus (2,11).…”