Decaprenyl diphosphate synthesis in Mycobacterium tuberculosis

Z-prenyl diphosphate synthases catalyze the sequential condensation of isopentenyl diphosphate with allylie diphosphates to synthesize polyprenyl diphosphates. In myeobaeteria, these are precursors of deeaprenyl phosphate, a molecule which plays a central role in the biosynthesis of essential myeobaeterial cell wall components, such as the mycolyl-arabinogalaetan-peptidoglyean complex and lipoarablnomannan. Recently, it was demonstrated that open reading frame Rv2361c of the Mycobacterium tuberculosis H37Rv genome encodes a unique prenyl diphosphate synthase (M. C. Schulbach, P. J. Brennan, and D. C. Crick, J. Biol. Chem. 275:22876-22881, 2000). We have now purified the enzyme to near homogeneity by using an Escherichia coli expression system and have shown that the product of this enzyme is decaprenyl diphosphate. Rv2361c has an absolute requirement for divalent cations and an optimal pH range of 7.5 to 8.5, and the activity is stimulated by both detergent and dithiothreitol. The enzyme catalyzes the addition of isopentenyl diphosphate to geranyl diphosphate, neryi diphosphate, [omega],E,E-farnesyl diphosphate, [omega],E,Z-farnesyl diphosphate, or [omega],E,E,E-geranylgeranyl diphosphate, with [K.sub.m] values for the allylic substrates of 490, 29, 84, 290, and 40 [mu]M, respectively. The [K.sub.m] value for isopentenyl diphosphate is 89 [mu]M. The catalytic efficiency is greatest when to, [omega]E,Z-farnesyl diphosphate is used as the allylic acceptor, suggesting that this is the natural substrate in vivo, a conclusion that is supported by previous structural studies of decaprenyl phosphoryl mannose isolated from M. tuberculosis. This is the first report of a bacterial Z-prenyl diphosphate synthase that preferentially utilizes an allylic diphosphate primer having the [alpha]-isoprene unit in the Z configuration, indicating that Rv1086 ([omega],E,Z-farnesyl diphosphate synthase) and Rv2361c act sequentially in the biosynthetic pathway that leads to the formation of decaprenyl phosphate in M. tuberculosis.