Publications

• The structural basis of chain length control in Rv1086.

  Wang W., Dong C., McNeil M., Kaur D., Mahapatra S., Crick D.C., Naismith J.H.

  In Mycobacterium tuberculosis, two related Z-prenyl diphosphate synthases, E,Z-farnesyl diphosphate synthase (Rv1086) and decaprenyl diphosphate synthase (Rv2361c), work in series to synthesize decaprenyl phosphate (C(50)) from isopentenyl diphosphate and E-geranyl diphosphate. Decaprenyl phosphat ... >> More

  In Mycobacterium tuberculosis, two related Z-prenyl diphosphate synthases, E,Z-farnesyl diphosphate synthase (Rv1086) and decaprenyl diphosphate synthase (Rv2361c), work in series to synthesize decaprenyl phosphate (C(50)) from isopentenyl diphosphate and E-geranyl diphosphate. Decaprenyl phosphate plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan; thus, its synthesis has attracted considerable interest as a potential therapeutic target. Rv1086 is a unique prenyl diphosphate synthase in that it adds only one isoprene unit to geranyl diphosphate, generating the 15-carbon product (E,Z-farnesyl diphosphate). Rv2361c then adds a further seven isoprene units to E,Z-farnesyl diphosphate in a processive manner to generate the 50-carbon prenyl diphosphate, which is then dephosphorylated to generate a carrier for activated sugars. The molecular basis for chain-length discrimination by Rv1086 during synthesis is unknown. We also report the structure of apo Rv1086 with citronellyl diphosphate bound and with the product mimic E,E-farnesyl diphosphate bound. We report the structures of Rv2361c in the apo form, with isopentenyl diphosphate bound and with a substrate analogue, citronellyl diphosphate. The structures confirm the enzymes are very closely related. Detailed comparison reveals structural differences that account for chain-length control in Rv1086. We have tested this hypothesis and have identified a double mutant of Rv1086 that makes a range of longer lipid chains. << Less

  J. Mol. Biol. 381:129-140(2008) [PubMed] [EuropePMC]

• Decaprenyl diphosphate synthesis in Mycobacterium tuberculosis.

  Kaur D., Brennan P.J., Crick D.C.

  Z-prenyl diphosphate synthases catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphates to synthesize polyprenyl diphosphates. In mycobacteria, these are precursors of decaprenyl phosphate, a molecule which plays a central role in the biosynthesis of essential myco ... >> More

  Z-prenyl diphosphate synthases catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphates to synthesize polyprenyl diphosphates. In mycobacteria, these are precursors of decaprenyl phosphate, a molecule which plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan. 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, neryl diphosphate, omega,E,E-farnesyl diphosphate, omega,E,Z-farnesyl diphosphate, or omega,E,E,E-geranylgeranyl diphosphate, with Km values for the allylic substrates of 490, 29, 84, 290, and 40 microM, respectively. The Km value for isopentenyl diphosphate is 89 microM. The catalytic efficiency is greatest when 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. << Less

  J. Bacteriol. 186:7564-7570(2004) [PubMed] [EuropePMC]

• Polyprenyl phosphate biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

  Crick D.C., Schulbach M.C., Zink E.E., Macchia M., Barontini S., Besra G.S., Brennan P.J.

  Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activitie ... >> More

  Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in M. tuberculosis are 10-to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in M. tuberculosis only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes omega,E,Z-farnesyl diphosphate and the other present in the cytosol that synthesizes omega,E,E,E-geranylgeranyl diphosphate. In M. smegmatis, the omega,E, Z-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the omega,E,E,E-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however, omega,E,E,E-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of M. smegmatis and M. tuberculosis can be attributed to distinct enzymatic differences between these two organisms. << Less

  J. Bacteriol. 182:5771-5778(2000) [PubMed] [EuropePMC]

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