decaprenyl-phosphate and arabinofuranose

Mycobacteria and corynebacteria use decaprenylphosphoryl-β-D-arabinofuranose (DPA) as a critical cell wall building block. Arabinofuranosyltransferases that process this substrate to mediate cell wall assembly have served as drug targets, but little is known about the substrate specificity of any of these enzymes. To probe substrate recognition of DPA, we developed a general and efficient synthetic route to β-D-arabinofuranosyl phosphodiesters. In this approach, the key glycosyl phosphodiester bond-forming reaction proceeds with high β-selectivity. In addition to its stereoselectivity, our route provides the means to readily access a variety of different lipid analogues, including aliphatic and polyprenyl substrates.

Topics: Arabinose; Cell Wall; Glycolipids; Glycosyltransferases; Lipids; Molecular Structure; Mycobacterium; Polyisoprenyl Phosphates; Stereoisomerism; Substrate Specificity

The major cell wall polysaccharide of mycobacteria is a branched-chain arabinogalactan in which arabinan chains are attached to the 5 carbon of some of the 6-linked galactofuranose residues; these arabinan chains are composed exclusively of D-arabinofuranose (Araf) residues. The immediate precursor of the polymerized Araf is decaprenylphosphoryl-D-Araf, which is derived from 5-phosphoribose 1-diphosphate (pRpp) in an undefined manner. On the basis of time course, feedback, and chemical reduction experiment results we propose that decaprenylphosphoryl-Araf is synthesized by the following sequence of events. (i) pRpp is transferred to a decaprenyl-phosphate molecule to form decaprenylphosphoryl-beta-D-5-phosphoribose. (ii) Decaprenylphosphoryl-beta-D-5-phosphoribose is dephosphorylated to form decaprenylphosphoryl-beta-D-ribose. (iii) The hydroxyl group at the 2 position of the ribose is oxidized and is likely to form decaprenylphosphoryl-2-keto-beta-D-erythro-pentofuranose. (iv) Decaprenylphosphoryl-2-keto-beta-D-erythro-pentofuranose is reduced to form decaprenylphosphoryl-beta-D-Araf. Thus, the epimerization of the ribosyl to an arabinosyl residue occurs at the lipid-linked level; this is the first report of an epimerase that utilizes a lipid-linked sugar as a substrate. On the basis of similarity to proteins implicated in the arabinosylation of the Azorhizobium caulidans nodulation factor, two genes were cloned from the Mycobacterium tuberculosis genome and expressed in a heterologous host, and the protein was purified. Together, these proteins (Rv3790 and Rv3791) are able to catalyze the epimerization, although neither protein individually is sufficient to support the activity.

Topics: Arabinose; Bacterial Proteins; Carbohydrate Epimerases; Diphosphates; Mycobacterium smegmatis; Polyisoprenyl Phosphates; Polysaccharides; Recombinant Proteins; Ribose