Purification, enzymatic characterization, and inhibition of the Z-farnesyl diphosphate synthase from Mycobacterium tuberculosis.
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).
Schulbach
M C
MC
Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523, USA.
Mahapatra
S
S
Macchia
M
M
Barontini
S
S
Papi
C
C
Minutolo
F
F
Bertini
S
S
Brennan
P J
PJ
Crick
D C
DC
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
D017931:DNA Primers; D004791:Enzyme Inhibitors; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase