Isopentenyl pyrophosphate synthesis in bacteria: Genes and enzymes of the mevalonate pathway.

摘要

Many bacteria employ the non-mevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, Gram-positive cocci and Borrelia burgdorferi use exclusively the mevalonate pathway, which is essential for their growth (Wilding, E. I., Kim, D-Y., Bryant, A. P., Gwynn, M. N., Lunsford, R. D., McDevitt, D., Myers, J. E., Jr., Rosenberg, M., Sylvester, D., Stauffacher, C. V., and Rodwell, V. W. 2000. Essentiality, expression and characterization of the Class II HMG-CoA reductase of Staphylococcus aureus. J. Bacteriol. 182:5147--5152). Enzymes of the mevalonate pathway thus are potential targets for drug intervention. The enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes 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. The mvaE gene was cloned from Enterococcus faecalis and expressed with an N-terminal histidine tag in Escherichia coli. The gene product was purified by nickel affinity chromatography and catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Optimal pH and temperature, DeltaHa, and Km values were determined for HMG-CoA reductase activity. A millimolar Ki for a statin drug confirmed that E. faecalis HMG-CoA reductase is a Class II enzyme. The oxidoreductant was NADP(H). Consistent with participation of a histidine during stage one of the HMG-CoA reductase reaction, diethylpyrocarbonate blocked formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases implicated this histidine as His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme. The mvaK1 gene encoding E. faecalis mevalonate kinase was PCR-cloned and expressed with a C-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. Temperature and pH optima, DeltaHa, and Km values were determined. Mevalonate kinase exhibits broad phosphoryl donor specificity. The K i for inhibition by ADP with respect to ATP was 2.7 mM. The characterization of the two bacterial enzymes of the mevalonate pathway is potentially important in the development of antibiotics against pathogens.