title: Synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate and kinetic studies of Mycobacterium tuberculosis IspF.
abstract: Many pathogenic bacteria utilize the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, two major building blocks of isoprenoid compounds. The fifth enzyme in the MEP pathway, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP) synthase (IspF), catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to ME-CPP with a corresponding release of cytidine 5-monophosphate (CMP). Because there is no ortholog of IspF in human cells, IspF is of interest as a potential drug target. However, study of IspF has been hindered by a lack of enantiopure CDP-ME2P. Herein, we report the first, to our knowledge, synthesis of enantiomerically pure CDP-ME2P from commercially available D-arabinose. Cloned, expressed, and purified M. tuberculosis IspF was able to utilize the synthetic CDP-ME2P as a substrate, a result confirmed by mass spectrometry. A convenient, sensitive, in vitro IspF assay was developed by coupling the CMP released during production of ME-CPP to mononucleotide kinase, which can be used for high throughput screening.
authors.affiliation: Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1682, USA. Praba@colostate.edu
authors.forename: Prabagaran
authors.initials: P
authors.lastname: Narayanasamy
authors.forename: Hyungjin
authors.initials: H
authors.lastname: Eoh
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Brennan
authors.forename: Dean C
authors.initials: DC
authors.lastname: Crick
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004896:Erythritol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013403:Sugar Phosphates / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology
chemical_list: C406803:4-diphosphocytidyl-2C methylerythritol 2-phosphate; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013403:Sugar Phosphates; D019761:Phosphorus-Oxygen Lyases; D004896:Erythritol