title: Characterization of the Mycobacterium tuberculosis 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase: potential for drug development.
abstract: Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-D-erythritol is formed from 2-C-methyl-D-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5'-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 microM for MEP and 53.2 microM for CTP. Calculated kcat and kcat/Km values were 0.72 min(-1) and 12.3 mM(-1) min(-1) for MEP and 1.0 min(-1) and 18.8 mM(-1) min(-1) for CTP, respectively.
authors.affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Hyungjin
authors.initials: H
authors.lastname: Eoh
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Brown
authors.forename: Lori
authors.initials: L
authors.lastname: Buetow
authors.forename: William N
authors.initials: WN
authors.lastname: Hunter
authors.forename: Tanya
authors.initials: T
authors.lastname: Parish
authors.forename: Devinder
authors.initials: D
authors.lastname: Kaur
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Brennan
authors.forename: Dean C
authors.initials: DC
authors.lastname: Crick
mesh_terms: D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000378:metabolism; D004795:Enzyme Stability; D004896:Erythritol / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020043:Genes, Essential; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism
chemical_list: C114232:2-C-methylerythritol 4-phosphate; C400182:4-diphosphocytidyl-2-C-methylerythritol; D002413:Cations, Divalent; D003067:Coenzymes; D011994:Recombinant Proteins; D013403:Sugar Phosphates; D003570:Cytidine Triphosphate; C400183:4-diphosphocytidyl-2-C--methylerythritol synthase; D009713:Nucleotidyltransferases; D004896:Erythritol