title: Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens.
abstract: Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.
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: Prabagaran
authors.initials: P
authors.lastname: Narayanasamy
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Brown
authors.forename: Tanya
authors.initials: T
authors.lastname: Parish
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; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004896:Erythritol / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D010770:Phosphotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013403:Sugar Phosphates / Q000737:chemistry / Q000378:metabolism
chemical_list: C114232:2-C-methylerythritol 4-phosphate; C400182:4-diphosphocytidyl-2-C-methylerythritol; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D013403:Sugar Phosphates; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C404502:IspE protein, E coli; D004896:Erythritol