The mevalonate pathway of isopentenyl pyrophosphate biosynthesis in Enterococcus faecalis: A potential target for antimicrobial agents

Autumn Lee Sutherlin, Purdue University

Abstract

Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multi-drug resistant strains of Gram-positive cocci employ the mevalonate pathway for IPP biosynthesis (E. I. Wilding et al. Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in Gram-positive cocci. J. Bacteriol. 182:4319, 2000). Bacterial mevalonate pathway enzymes therefore offer potential targets for development of inhibitors for use as antibiotics. Enterococci possess an open reading frame, mvaE, that appears to encode two catalytic centers of the mevalonate pathway, acetoacetyl coenzyme A thiolase and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium and Enterococcus hirae. mvaE was cloned from Enterococcus faecalis and expressed with an N-terminal histidine tag in Escherichia coli. The 86.5 kDa mvaE gene product, purified by nickel affinity chromatography, catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, ΔH a, Km values and pH optima were determined for acetoacetyl-CoA thiolase. Kinetic studies implicated a ping-pong mechanism. Coenzyme A inhibited competitively with acetyl-CoA. The polymerase chain reaction and E. faecalis genomic DNA were used to isolate the mvaS gene that encodes HMG-CoA synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in E. coli with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni2+-agarose to apparent homogeneity (specific activity 10 μmoles/min/mg). The enzyme is a dimer, mass 83.9 kDa, S20w 5.3. Optimal activity occurred at pH 9.8 in 2.0 mM MgCl2 at 37°C. ΔHa was 6000 calories per mole. The stoichiometry per monomer of acetyl-CoA binding was 1.2 ± 0.2 and of covalent acetylation was 0.60 ± 0.02. Km for the hydrolysis of acetyl-CoA was 10 μM. Coupled conversion of acetyl-CoA to mevalonate was achieved using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis. A hollow fiber reactor and NiNTA affinity support were used to trap the fusion protein, HMG-CoA synthase, and unfused HMG-CoA reductase from E. faecalis to form a bioreactor that converted acetyl-CoA to mevalonate.

Degree

Ph.D.

Advisors

Rodwell, Purdue University.

Subject Area

Biochemistry

COinS