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, ,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 ,E-geranyl diphosphate or ,Zneryl diphosphate yielding ,E,Z-farnesyl diphosphate and ,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 M for isopentenyl diphosphate, 38 M for geranyl diphosphate, and 16 M for neryl diphosphate. Inhibitors of the Zfarnesyl diphosphate synthase were designed and chemically synthesized as stable analogs of ,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).Isoprenyl diphosphate synthases catalyze the condensation of an allylic diphosphate with isopentenyl diphosphate (IPP, 1 C 5 ) via an electrophilic alkylation reaction to produce longer allylic diphosphates (1, 2). Chain elongation continues until a physiologically appropriate chain length is reached, at which time the molecule may undergo further modifications (dephosphorylation, cyclization, or head-to-head condensation reactions). Polyprenyl phosphate (Pol-P) is formed by dephosphorylation of an allylic prenyl diphosphate chain. The predominant form of prokaryotic Pol-P is ,diE,polyZ-undecaprenyl phosphate 2 (C 55 ); however, there are documented exceptions in Paracoccus denitrificans (3) and in Mycobacterium sp. (4 -8). M. smegmatis contains heptaprenyl diphosphate (8) (C 35 , four saturated, three Z double bonds) and decaprenyl diphosphate (5) (C 50 , , one E, and eight Z double bonds), whereas M. tuberculosis contains only decaprenyl phosphate (6). Although the stereochemistry of decaprenyl phosphate from M. tuberculosis has not been determined, our enzymatic studies suggest that it has similar stereochemistry to decaprenyl phosphate from M. smegmatis (9).Pol-P is central to prokaryotic cell wall synthesis as a sugar carrier, and it has been reported that the levels of Pol-P may be rate-limiting for in vivo cell wall synthesis (10 -13). Our laboratory has shown that Pol-P is instrumental in the synthesis of each component of the covalently linked peptidoglycan-arabinogalactan-mycolic acid cell wall core of mycobacteria, and other noncovalently associated macromolecules such as lipomannan and lipoarabinomannan (5,14,15). The importance of Pol-P is also demons...