Identification and Active Expression of the Mycobacterium tuberculosis Gene Encoding 5-Phospho-α-D-ribose-1-diphosphate: Decaprenyl-phosphate 5-Phosphoribosyltransferase, the First Enzyme Committed to Decaprenylphosphoryl-D-arabinose Synthesis

Abstract

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-␣-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-␣-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.The cell wall core of Mycobacterium tuberculosis consists of an inner peptidoglycan layer and an outer lipid mycolic acid layer (1). These two layers are connected by the polysaccharide arabinogalactan (1). The structure of arabinogalactan is conveniently divided into three regions, namely the linker region (2), a D-galactofuranosyl region (3), and an arabinofuranosyl region (3). Enzymes required for arabinogalactan formation have been shown to be essential for mycobacterial growth (4, 5). The donor for the arabinofuranosyl residues has been shown to be decaprenylphosphoryl-D-arabinose (6 -8). The carbon atoms of the arabinosyl residue come originally from the pentose shunt (9) and specifically from phosphoribose diphosphate (pRpp) 1 (10). The first reaction in the formation of decaprenylphosphoryl-Darabinose is the transfer of ribose-5-phosphate from pRpp to decaprenylphosphate 2 (10) to form decaprenylphosphoryl-5-phosphoribose (DPPR). Genetic analysis of a gene cluster found in Azorhizobium caulinodans responsible for D-arabinosylation of nod factor 3 (11) revealed four open reading frames; three of these have homology to M. tuberculosis genes. Rv3806c, which shows homology to the noeC gene of A. caulinodans, was hypothesized to be the 5-phospho-␣-D-ribose-1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase gene because of its transmembrane d...

Identification of a Novel Arabinofuranosyltransferase AftB Involved in a Terminal Step of Cell Wall Arabinan Biosynthesis in Corynebacterianeae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis

“…1A), which is located in close proximity to the antigen 85 complex-encoding genes fbpA and fbpD (36). Furthermore, Rv3805c is likely to form an operon together with ubiA, which is required for prenyl transfer to 5-phosphoribose pyrophosphate to form decaprenylphosphoryl-5-phosphoribose before conversion to DPA (27,28) and glfT, which is responsible for establishing the galactan backbone of AG (20,21). The apparent fundamental function of aftB is indicated by the fact that the genome organization of this particular region is syntenic in Corynebacterianeae, including all Mycobacterium and Corynebacterium species analyzed to date (see Fig.…”

“…Complementation of C. glutamicum⌬aftB with either pMSX-Cg-aftB or pMSX-Mt-aftB restored the mutant to a wild type phenotype. For the purpose of significance, C. glutamicum⌬aftB comple- (26,27) and the known galactofuranosyltransferase glfT (20,21) and UDP-Galp mutase enzyme glf (45). Downstream of aftB the genes fbpA and fbpD are located which encode mycolyltransferases for decoration of the terminal arabinose residues with mycolic acids (6,36).…”

Deletion of Cg-emb in Corynebacterianeae Leads to a Novel Truncated Cell Wall Arabinogalactan, whereas Inactivation of Cg-ubiA Results in an Arabinan-deficient Mutant with a Cell Wall Galactan Core

“…1B). The orthologue of M. tuberculosis Rv3806c in C. glutamicum is NCgl2781, and during compilation of this report was shown biochemically to perform the first step of DPA biosynthesis producing decaprenylphosphoryl-5-phosphoribose from 5-phosphoribofuranose pyrophosphate and decaprenol phosphate (39). To inactivate ubiA of C. glutamicum, plasmid pCg::ubiA was constructed and C. glutamicum transformed to kanamycin resistance.…”

Prokaryotic Expression, Identification and Bioinformatics Analysis of the Mycobacterium tuberculosis Rv3807c Gene Encoding the Putative Enzyme Committed to Decaprenylphosphoryl-d-arabinose Synthesis

“…Decaprenylphosphoryl-D-arabinose is synthesized initially from the transfer of phosphoribosyl pyrophosphate (PRPP) to decaprenyl phosphate (DP) to form 5-phosphodecaprenylphospho-ribose (5-p-DPR) via the 5-phosphorobosyl transferase (Rv3806c) [9]. Subsequently, the 5-phosphate unit is removed from 5-phospho-decaprenylphospho-ribose to form decaprenyl-phosphoryl-ribose (DPR) by the putative Rv3807 phospholipid phosphatase [10,11].…”