Purification, enzymatic characterization, and inhibition of the Z-farnesyldiphosphate 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 s ynthase. The product of this enzyme, omega ,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a centra l role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinoma nnan. We have now purified Z-farnesyl diphosphate synthase to near homogene ity using a novel mycobacterial expression system. Z-Farnesyl diphosphate s ynthase catalyzed the addition of isopentenyl diphosphate to omega ,E-geran yl diphosphate or omega ,Z-neryl diphosphate yielding omega ,E,Z-farnesyl d iphosphate and omega ,Z,Z-farnesyl diphosphate, respectively. The enzyme ha s an absolute requirement for a divalent cation, an optimal pH range of 7-8 , and K-m values of 124 muM for isopentenyl diphosphate, 38 muM for geranyl diphosphate, and 16 muM for neryl diphosphate. Inhibitors of the Z-farnesy l diphosphate synthase were designed and chemically synthesized as stable a nalogs of omega ,E-geranyl diphosphate in which the labile diphosphate moie ty was replaced with stable moieties. Studies with these compounds revealed that the active site of Z-farnesyl diphosphate synthase differs substantia lly from E-farnesyl diphosphate synthase from pig brain (Sus scrofa).