Decaprenyl diphosphate synthesis in Mycobacterium tuberculosis.
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.
Kaur
Devinder
D
Mycobacterial Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.
Brennan
Patrick J
PJ
Crick
Dean C
DC
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis* / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011106:Polyisoprenyl Phosphates; C068697:decaprenyl phosphate; D019883:Alkyl and Aryl Transferases; C412569:Rv1086 protein, Mycobacterium tuberculosis; C499606:Rv2361c protein, Mycobacterium tuberculosis; C039821:decaprenyl pyrophosphate synthetase