1887

Abstract

Decaprenylphosphoryl--arabinose (DPA) has been shown to be the donor of the essential -arabinofuranosyl residues found in the cell wall of . DPA is formed from phosphoribose diphosphate in a four-step process. The first step is the nucleophilic replacement of the diphosphate group with decaprenyl phosphate. This reaction is catalysed by the integral membrane protein 5-phospho---ribose-1-diphosphate : decaprenyl-phosphate 5-phosphoribosyltransferase (DPPR synthase). The enzyme is essential for growth and thereby an important target candidate for the development of new tuberculosis drugs. Although membrane proteins are an important subset of targets for current antibacterial agents, details about the structures and the active sites of such proteins are often not readily available by X-ray crystallography. To begin a different approach to the issue, homologues from and were expressed in and shown to be active DPPR synthases. This was followed by bioinformatic analyses of the aligned sequences and then by site-directed mutagenesis of amino acids identified as likely to be important for activity. The results suggested that the enzymic synthesis of decaprenyl-phosphate 5-phosphoribose (DPPR) occurs on the cytoplasmic side of the plasma membrane. Amino acid substitutions showed that the predicted cytoplasmic N-terminal region and two cytoplasmic loops are involved in substrate binding and/or catalysis along with parts of some adjoining inner membrane regions. The enzyme lacks the classical phosphoribose diphosphate (pRpp) binding site found in nucleic acid precursor enzymes of both prokaryotes and eukaryotes but instead contains a conserved NDxxD motif required for enzymic activity. Thus, it is plausible that this DPPR synthase has a pRpp binding site that is different from that of the classical eukaryotic enzymes, and further work to develop inhibitors against this enzyme is thereby encouraged.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/013532-0
2008-03-01
2019-10-14
Loading full text...

Full text loading...

/deliver/fulltext/micro/154/3/736.html?itemId=/content/journal/micro/10.1099/mic.0.2007/013532-0&mimeType=html&fmt=ahah

References

  1. Brauer, L., Brandt, W. & Wessjohann, L. A. ( 2004; ). Modeling the E. coli 4-hydroxybenzoic acid oligoprenyltransferase (ubiA transferase) and characterization of potential active sites. J Mol Model 10, 317–327.[CrossRef]
    [Google Scholar]
  2. Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J., Higgins, D. G. & Thompson, J. D. ( 2003; ). Multiple sequence alignment with the clustal series of programs. Nucleic Acids Res 31, 3497–3500.[CrossRef]
    [Google Scholar]
  3. Craig, S. P. & Eakin, A. E. ( 2000; ). Purine phosphoribosyltransferases. J Biol Chem 275, 20231–20234.[CrossRef]
    [Google Scholar]
  4. Dover, L. G., Cerdeno-Tarraga, A. M., Pallen, M. J., Parkhill, J. & Besra, G. S. ( 2004; ). Comparative cell wall core biosynthesis in the mycolated pathogens, Mycobacterium tuberculosis and Corynebacterium diphtheriae. FEMS Microbiol Rev 28, 225–250.[CrossRef]
    [Google Scholar]
  5. Hemmi, H., Shibuya, K., Takahashi, Y., Nakayama, T. & Nishino, T. ( 2004; ). (S)-2,3-Di-O-geranylgeranylglyceryl phosphate synthase from the thermoacidophilic Archaeon Sulfolobus solfataricus – molecular cloning and characterization of a membrane-intrinsic prenyltransferase involved in the biosynthesis of archaeal ether-linked membrane lipids. J Biol Chem 279, 50197–50203.[CrossRef]
    [Google Scholar]
  6. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. & Pease, L. R. ( 1989; ). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59.[CrossRef]
    [Google Scholar]
  7. Huang, H., Scherman, M. S., D'Haeze, W., Vereecke, D., Holsters, M., Crick, D. C. & McNeil, M. R. ( 2005; ). Identification and active expression of the M. tuberculosis gene encoding, 5-phospho-α-d-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase, the first enzyme committed to decaprenylphosphoryl-d-arabinose synthesis. J Biol Chem 280, 24539–24543.[CrossRef]
    [Google Scholar]
  8. Kall, L., Krogh, A. & Sonnhammer, E. L. ( 2004; ). A combined transmembrane topology and signal peptide prediction method. J Mol Biol 338, 1027–1036.[CrossRef]
    [Google Scholar]
  9. Kantardjieff, K. A., Vasquez, C., Castro, P., Warfel, N. M., Rho, B. S., Lekin, T., Kim, C. Y., Segelke, B. W., Terwilliger, T. C. & Rupp, B. ( 2005; ). Structure of pyrR (Rv1379) from Mycobacterium tuberculosis: a persistence gene and protein drug target. Acta Crystallogr D Biol Crystallogr 61, 355–364.
    [Google Scholar]
  10. Leatherbarrow, R. J. ( 2001; ). GraFit Version 5, 5th edn. Horley, UK: Erithacus Software Ltd.
  11. Mergaert, P., D'Haeze, W., Fernandez-Lopez, M., Geelen, D., Goethals, K., Prome, J. C., Van Montagu, M. & Holsters, M. ( 1996; ). Fucosylation and arabinosylation of Nod factors in Azorhizobium caulinodans: involvement of nolK, nodZ as well as noeC and/or downstream genes. Mol Microbiol 21, 409–419.[CrossRef]
    [Google Scholar]
  12. Mikusova, K., Huang, H., Yagi, T., Holsters, M., Vereecke, D., D'Haeze, W., Scherman, M. S., Brennan, P. J., McNeil, M. R. & Crick, D. C. ( 2005; ). Decaprenylphosphoryl arabinofuranose, the donor of the d-arabinofuranosyl residues of mycobacterial arabinan, is formed via a two-step epimerization of decaprenylphosphoryl ribose. J Bacteriol 187, 8020–8025.[CrossRef]
    [Google Scholar]
  13. Saiki, K., Mogi, T., Hori, H., Tsubaki, M. & Anraku, Y. ( 1993; ). Identification of the functional domains in heme O synthase. Site-directed mutagenesis studies on the cyoE gene of the cytochrome bo operon in Escherichia coli. J Biol Chem 268, 26927–26934.
    [Google Scholar]
  14. Scherman, M., Weston, A., Duncan, K., Whittington, A., Upton, R., Deng, L., Comber, R., Friedrich, J. D. & McNeil, M. ( 1995; ). The biosynthetic origin of the mycobacterial cell wall arabinosyl residues. J Bacteriol 177, 7125–7130.
    [Google Scholar]
  15. Scherman, M. S., Kalbe-Bournonville, L., Bush, D., Xin, Y. & McNeil, M. ( 1996; ). Polyprenylphosphate-pentoses in mycobacteria are synthesized from phosphoribose pyrophosphate. J Biol Chem 271, 29652–29658.[CrossRef]
    [Google Scholar]
  16. Wang, K. & Ohnuma, S. ( 1999; ). Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecular evolution. Trends Biochem Sci 24, 445–451.[CrossRef]
    [Google Scholar]
  17. Wolucka, B. A., McNeil, M. R., de Hoffmann, E., Chojnacki, T. & Brennan, P. J. ( 1994; ). Recognition of the lipid intermediate for arabinogalactan/arabinomannan biosynthesis and its relation to the mode of action of ethambutol on mycobacteria. J Biol Chem 269, 23328–23335.
    [Google Scholar]
  18. Xin, Y., Lee, R. E., Scherman, M. S., Khoo, K. H., Besra, G. S., Brennan, P. J. & McNeil, M. ( 1997; ). Characterization of the in vitro synthesized arabinan of mycobacterial cell walls. Biochim Biophys Acta 1335, 231–234.[CrossRef]
    [Google Scholar]
  19. Zhou, G. P. & Troy, F. A. ( 2005; ). NMR study of the preferred membrane orientation of polyisoprenols (dolichol) and the impact of their complex with polyisoprenyl recognition sequence peptides on membrane structure. Glycobiology 15, 347–359.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/013532-0
Loading
/content/journal/micro/10.1099/mic.0.2007/013532-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error