title: 2C-Methyl-d-erythritol 4-phosphate enhances and sustains cyclodiphosphate synthase IspF activity.
abstract: There is significant progress toward understanding catalysis throughout the essential MEP pathway to isoprenoids in human pathogens; however, little is known about pathway regulation. The present study begins by testing the hypothesis that isoprenoid biosynthesis is regulated via feedback inhibition of the fifth enzyme cyclodiphosphate synthase IspF by downstream isoprenoid diphosphates. Here, we demonstrate recombinant E. coli IspF is not inhibited by downstream metabolites isopentenyl diphosphate (IDP), dimethylallyl diphosphate (DMADP), geranyl diphosphate (GDP), and farnesyl diphosphate (FDP) under standard assay conditions. However, 2C-methyl-d-erythritol 4-phosphate (MEP), the product of reductoisomerase IspC and first committed MEP pathway intermediate, activates and sustains this enhanced IspF activity, and the IspF-MEP complex is inhibited by FDP. We further show that the methylerythritol scaffold itself, which is unique to this pathway, drives the activation and stabilization of active IspF. Our results suggest a novel feed-forward regulatory mechanism for 2C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) production and support an isoprenoid biosynthesis regulatory mechanism via feedback inhibition of the IspF-MEP complex by FDP. The results have important implications for development of inhibitors against the IspF-MEP complex, which may be the physiologically relevant form of the enzyme.
authors: Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
authors: J Kipchirchir
authors: JK
authors: Bitok
authors: Caren Freel
authors: CF
authors: Meyers
mesh_terms: D002384:Catalysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004896:Erythritol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D007700:Kinetics; D019761:Phosphorus-Oxygen Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D012717:Sesquiterpenes / Q000494:pharmacology; D013403:Sugar Phosphates / Q000494:pharmacology*; D013729:Terpenes / Q000378:metabolism
chemical_list: C114232:2-C-methylerythritol 4-phosphate; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D013403:Sugar Phosphates; D013729:Terpenes; D006153:Guanosine Diphosphate; C004808:farnesyl pyrophosphate; D019761:Phosphorus-Oxygen Lyases; C400181:ISPF protein, E coli; D004896:Erythritol