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Polyprenyl phosphates (Pol-P) are involved in the biosynthesis of bacterial cell walls (14), and their availability is rate limiting for several aspects of cell wall synthesis in Staphylococcus aureus (15) and Bacillus spp. (2). It has also been suggested that the rate of synthesis of lipid I (in peptidoglycan synthesis) of Escherichia coli may be dependent on the pool level of Pol-P (26), and Baddiley (4) reported that Pol-P levels could regulate the rate of bacterial cell wall synthesis in vivo. Mycobacterium smegmatis is known to contain two forms of Pol-P that are covalently attached to mannose (25). These are structurally unusual in that the decaprenyl phosphate contains one ω-isoprene unit, one E-isoprene unit, and eight Z-isoprene units (mono-E, poly-Z) (28) and the heptaprenyl phosphate consists of either four saturated isoprene units on the omega end of the molecule, two E-isoprene units, and one Z-isoprene unit (6) or four saturated and three Z-isoprene units (27) (Fig. ​(Fig.1A)1A) (the stereochemical configuration of the isoprene units is always listed starting at the omega end of the molecule). It appears that Mycobacterium tuberculosis may be more typical than M. smegmatis, as a single predominant Pol-P (decaprenyl phosphate) was identified in this species, however; the stereochemistries of the individual isoprene units were not determined (24). FIG. 1 Structures of heptaprenyl diphosphate and decaprenyl diphosphate (A) and prenyl diphosphate synthesis scheme (B). The structure of the heptaprenyl diphosphate is drawn as described for heptaprenyl phosphoryl mannose by Wolucka and de Hoffmann (27). The ... The fact that both forms of Pol-P in M. smegmatis are glycosylated suggested that both could be involved in the synthesis of cell wall polysaccharides. Our laboratory has shown that M. smegmatis utilizes its unusual Pol-P molecules in many stages of cell wall biosynthesis. Mature mycolic acids appear to be formed from precursors while attached to a heptaprenyl phosphate molecule (6). Decaprenyl-P-arabinose is a precursor of the arabinan portions of arabinogalactan, arabinomannan, and lipoarabinomannan (28). A polyprenyl diphosphate carrier lipid has been implicated in the synthesis of the linker unit galactan of M. smegmatis (17) and in the synthesis of linear forms of lipoarabinomannan (5). Despite the crucial role of Pol-P in bacterial cell wall biogenesis, little is known about its biosynthesis, especially in Mycobacterium spp. Pol-P is typically synthesized by enzymes that catalyze the 1′-4 condensations of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates (reaction primers) in order to generate longer, physiologically appropriate, allylic prenyl diphosphates (Fig. ​(Fig.1B).1B). The diphosphates are subsequently dephosphorylated to form the appropriate Pol-P. The importance of this biosynthetic pathway in mycobacterial biology is demonstrated by the fact that all species of mycobacteria tested (reference 19 and our unpublished data) are susceptible to the antibiotic bacitracin, which specifically binds prenyl diphosphate intermediates in Pol-P synthesis (22). Prenyl diphosphate synthases are very widespread in nature, but of the hundreds in existence, only a few have been studied (20). Thus, Pol-P synthesis is clearly important in the rate of bacterial growth and the synthesis of cell wall components essential for the viability of mycobacteria. The intriguing structural differences in Pol-P from M. smegmatis and M. tuberculosis prompted us to initiate an investigation of the biosynthesis of Pol-P in these two species to explain the enzymatic basis of these observations.