Identification and active expression of the Mycobacterium tuberculosis gene encoding 5-phospho-{alpha}-d-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase, the first enzyme committed to decaprenylphosphoryl-d-arabinose synthesis.
Decaprenylphosphoryl-d-arabinose, the lipid donor of mycobacterial d-arabinofuranosyl residues, is synthesized from phosphoribose diphosphate rather than from a sugar nucleotide. The first committed step in the process is the transfer of a 5-phosphoribosyl residue from phosphoribose diphosphate to decaprenyl phosphate to form decaprenylphosphoryl-5-phosphoribose via a 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phospho-ribosyltransferase. A candidate for the gene encoding this enzyme (Rv3806c) was identified in Mycobacterium tuberculosis, primarily via its homology to one of four genes responsible for d-arabinosylation of nodulation factor in Azorhizobium caulinodans. The resulting protein was predicted to contain eight or nine transmembrane domains. The gene was expressed in Escherichia coli, and membranes from the expression strain of E. coli but not from a control strain of E. coli were shown to convert phosphoribose diphosphate and decaprenyl phosphate into decaprenylphosphoryl-5-phosphoribose. Neither UDP-galactose nor GDP-mannose was active as a sugar donor. The enzyme favored polyprenyl phosphate with 50-60 carbon atoms, was unable to use C-20 polyprenyl phosphate, and used C-75 polyprenyl phosphate less efficiently than C-50 or C-60. It requires CHAPS detergent and Mg(2+) for activity. The Rv3806c gene encoding 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase is known to be essential for the growth of M. tuberculosis, and the tuberculosis drug ethambutol inhibits other steps in arabinan biosynthesis. Thus the Rv3806c-encoded enzyme appears to be a good target for the development of new tuberculosis drugs.
Huang
Hairong
H
Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523-1682, USA.
Scherman
Michael S
MS
D'Haeze
Wim
W
Vereecke
Danny
D
Holsters
Marcelle
M
Crick
Dean C
DC
McNeil
Michael R
MR
D000995:Antitubercular Agents / Q000494:pharmacology; D001089:Arabinose / Q000737:chemistry*; D020599:Azorhizobium caulinodans / Q000201:enzymology; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D002793:Cholic Acids / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D004977:Ethambutol / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000737:chemistry; D006155:Guanosine Diphosphate Mannose / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D010710:Phosphates / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012266:Ribose / Q000737:chemistry; D012268:Ribose-Phosphate Pyrophosphokinase / Q000737:chemistry*; D012267:Ribosemonophosphates / Q000737:chemistry*; D016622:Silver Staining; D013379:Substrate Specificity; D013997:Time Factors; D014530:Uridine Diphosphate / Q000737:chemistry; D014531:Uridine Diphosphate Galactose / Q000737:chemistry
D000995:Antitubercular Agents; D002793:Cholic Acids; D017931:DNA Primers; D003902:Detergents; D010710:Phosphates; D011134:Polysaccharides; D012267:Ribosemonophosphates; C030080:araban; D006153:Guanosine Diphosphate; D014531:Uridine Diphosphate Galactose; D006155:Guanosine Diphosphate Mannose; C031626:ribose-5-phosphate; D014530:Uridine Diphosphate; D012266:Ribose; D004977:Ethambutol; D001089:Arabinose; D012268:Ribose-Phosphate Pyrophosphokinase; D008274:Magnesium; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate