title: Purification, enzymatic characterization, and inhibition of the Z-farnesyl diphosphate synthase from Mycobacterium tuberculosis.
abstract: We have recently shown that open reading frame Rv1086 of the Mycobacterium tuberculosis H37Rv genome sequence encodes a unique isoprenyl diphosphate synthase. The product of this enzyme, omega,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinomannan. We have now purified Z-farnesyl diphosphate synthase to near homogeneity using a novel mycobacterial expression system. Z-Farnesyl diphosphate synthase catalyzed the addition of isopentenyl diphosphate to omega,E-geranyl diphosphate or omega,Z-neryl diphosphate yielding omega,E,Z-farnesyl diphosphate and omega,Z,Z-farnesyl diphosphate, respectively. The enzyme has an absolute requirement for a divalent cation, an optimal pH range of 7-8, and K(m) values of 124 micrometer for isopentenyl diphosphate, 38 micrometer for geranyl diphosphate, and 16 micrometer for neryl diphosphate. Inhibitors of the Z-farnesyl diphosphate synthase were designed and chemically synthesized as stable analogs of omega,E-geranyl diphosphate in which the labile diphosphate moiety was replaced with stable moieties. Studies with these compounds revealed that the active site of Z-farnesyl diphosphate synthase differs substantially from E-farnesyl diphosphate synthase from pig brain (Sus scrofa).
authors.affiliation: Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.
authors.forename: M C
authors.initials: MC
authors.lastname: Schulbach
authors.forename: S
authors.initials: S
authors.lastname: Mahapatra
authors.forename: M
authors.initials: M
authors.lastname: Macchia
authors.forename: S
authors.initials: S
authors.lastname: Barontini
authors.forename: C
authors.initials: C
authors.lastname: Papi
authors.forename: F
authors.initials: F
authors.lastname: Minutolo
authors.forename: S
authors.initials: S
authors.lastname: Bertini
authors.forename: P J
authors.initials: PJ
authors.lastname: Brennan
authors.forename: D C
authors.initials: DC
authors.lastname: Crick
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051228:Geranyltranstransferase; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011487:Protein Conformation; D013379:Substrate Specificity; D013552:Swine
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase