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Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A synthase, an enzyme of isopentenyl diphosphate
Autumn Sutherlin1, Matija Hedl, Barbara Sanchez-Neri
1Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Abstract:
Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni(2+)-agarose to apparent homogeneity and a specific activity of 10 micromol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s(20,w), 5.3). Optimal activity occurred in 2.0 mM MgCl(2) at 37(o)C. The DeltaH(a) was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK(a) of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 +/- 0.2 and that of covalent acetylation was 0.60 +/- 0.02. The K(m) for the hydrolysis of acetyl-CoA was 10 microM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis.
Insights
Multidrug-resistant bacteria use the mevalonate pathway for essential isopentenyl diphosphate (IPP) biosynthesis. Researchers isolated and characterized the mvaS gene encoding HMG-CoA synthase, a key enzyme in this pathway, identifying it as a potential antibiotic target.
Area of Science:
- Biochemistry
- Microbiology
- Drug Discovery
Background:
- Isopentenyl diphosphate (IPP) is crucial for isoprenoid biosynthesis.
- Multidrug-resistant gram-positive cocci exclusively utilize the mevalonate pathway for IPP production.
- Bacterial mevalonate pathway enzymes are potential targets for novel antibiotic development.
Purpose of the Study:
- To isolate and characterize the mvaS gene encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase from Enterococcus faecalis.
- To investigate the properties of the purified HMG-CoA synthase enzyme.
- To assess the potential of this enzyme as an antibiotic target.
Main Methods:
- Polymerase chain reaction (PCR) amplification of the mvaS gene from Enterococcus faecalis genomic DNA.
- Expression of the mvaS gene in Escherichia coli using a pET28 vector with an N-terminal histidine tag.
- Purification of the enzyme via Ni(2+)-agarose affinity chromatography.
- Enzyme characterization including analytical ultracentrifugation, optimal activity determination, and kinetic analysis (K(m)).
Main Results:
- The mvaS gene was successfully isolated and the encoded HMG-CoA synthase enzyme was expressed and purified to homogeneity.
- The enzyme exists as a dimer (83.9 kDa) and exhibits optimal activity at 37°C in 2.0 mM MgCl(2) and pH 9.8.
- Kinetic analysis revealed a K(m) of 10 μM for acetyl-CoA hydrolysis, with demonstrated coupled conversion to mevalonate.
Conclusions:
- The characterization of Enterococcus faecalis HMG-CoA synthase provides a foundation for structure-based drug design.
- The enzyme's essential role in the mevalonate pathway of resistant bacteria highlights its potential as a novel antibiotic target.
- Inhibiting this enzyme could lead to the development of new strategies against multidrug-resistant infections.