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Journal of Bacteriology, November 2004, p. 7564-7570, Vol. 186, No. 22
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.22.7564-7570.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Decaprenyl Diphosphate Synthesis in Mycobacterium tuberculosis

Devinder Kaur, Patrick J. Brennan, and Dean C. Crick^*

Mycobacterial Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado

Received 13 April 2004/ Accepted 12 August 2004

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, ,E,E-farnesyl diphosphate, ,E,Z-farnesyl diphosphate, or ,E,E,E-geranylgeranyl diphosphate, with K_m values for the allylic substrates of 490, 29, 84, 290, and 40 µM, respectively. The K_m value for isopentenyl diphosphate is 89 µM. The catalytic efficiency is greatest when ,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 -isoprene unit in the Z configuration, indicating that Rv1086 (,E,Z-farnesyl diphosphate synthase) and Rv2361c act sequentially in the biosynthetic pathway that leads to the formation of decaprenyl phosphate in M. tuberculosis.

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* Corresponding author. Mailing address: Department of Microbiology, Immunology, and Pathology, Colorado State University, 1682 Campus Delivery, Fort Collins, CO 80523-1682. Phone: (970) 491-3308. Fax: (970) 491-1815. E-mail: Dean.Crick{at}colostate.edu.

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Journal of Bacteriology, November 2004, p. 7564-7570, Vol. 186, No. 22
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.22.7564-7570.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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