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

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, ΔHa, 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, ΔHa, 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

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 μmole/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°C. ΔHa was 22 kcal/mole. Kinetic analysis suggested that the reaction proceeds via a sequential mechanism. Km values were 0.33 mM (mevalonate), 1.1 mM (ATP), and 3.3 mM (Mg2+). 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 Ki of 2.7 mM

IRF5 and IRF5 Disease-Risk Variants Increase Glycolysis and Human M1 Macrophage Polarization by Regulating Proximal Signaling and Akt2 Activation

Interferon regulatory factor 5 (IRF5) regulates inflammatory M1 macrophage polarization, and disease-associated IRF5 genetic variants regulate pattern-recognition-receptor (PRR)-induced cytokines. PRR-stimulated macrophages and M1 macrophages exhibit enhanced glycolysis, a central mediator of inflammation. We find that IRF5 is needed for PRR-enhanced glycolysis in human macrophages and in mice in vivo. Upon stimulation of the PRR nucleotide binding oligomerization domain containing 2 (NOD2) in human macrophages, IRF5 binds RIP2, IRAK1, and TRAF6. IRF5, in turn, is required for optimal Akt2 activation, which increases expression of glycolytic pathway genes and HIF1A as well as pro-inflammatory cytokines and M1 polarization. Furthermore, pro-inflammatory cytokines and glycolytic pathways co-regulate each other. Rs2004640/rs2280714 TT/TT IRF5 disease-risk-carrier cells demonstrate increased IRF5 expression and increased PRR-induced Akt2 activation, glycolysis, pro-inflammatory cytokines, and M1 polarization relative to GG/CC carrier macrophages. Our findings identify that IRF5 disease-associated polymorphisms regulate diverse immunological and metabolic outcomes and provide further insight into mechanisms contributing to the increasingly recognized important role for glycolysis in inflammation