Enterococcus faecalis mevalonate kinase.
Gram-positive pathogens synthesize isopentenyl diphosphate, the five-carbon precursor of isoprenoids, via the mevalonate pathway. The enzymes of this pathway are essential for the survival of these organisms, and thus may represent possible targets for drug design. To extend our investigation of the mevalonate pathway in Enterococcus faecalis, we PCR-amplified and cloned into pET-28b the mvaK1 gene thought to encode mevalonate kinase, the fourth enzyme of the pathway. Following transformation of the construct EFK1-pET28b into Escherichia coli BL21(DE3) cells, the expressed C-terminally hexahistidine-tagged protein was purified on a nickel affinity support to apparent homogeneity. The purified protein catalyzed the divalent ion-dependent phosphorylation of mevalonate to mevalonate 5-phosphate. The specific activity of the purified kinase was 24 micromole/min/mg protein. Based on sedimentation velocity data, E. faecalis mevalonate kinase exists in solution primarily as a monomer with a mass of 32.2 kD. Optimal activity occurred at pH 10 and at 37 degrees C. Delta H(a) was 22 kcal/mole. Kinetic analysis suggested that the reaction proceeds via a sequential mechanism. K(m) values were 0.33 mM (mevalonate), 1.1 mM (ATP), and 3.3 mM (Mg(2+)). Unlike mammalian mevalonate kinases, E. faecalis mevalonate kinase utilized all tested nucleoside triphosphates as phosphoryl donors. ADP, but not AMP, inhibited the reaction with a K(i) of 2.7 mM.
Hedl
Matija
M
Department of Biochemistry, Purdue University, West Lafayette, IN 47907-2063, USA.
Rodwell
Victor W
VW
D000244:Adenosine Diphosphate / Q000737:chemistry; D000249:Adenosine Monophosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008798:Mevalonic Acid / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014461:Ultracentrifugation
D001426:Bacterial Proteins; D002413:Cations, Divalent; D009711:Nucleotides; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D008798:Mevalonic Acid