Biosynthetic origin of mycobacterial cell wall arabinosyl residues

Abstract

Designing new drugs that inhibit the biosynthesis of the D-arabinan moiety of the mycobacterial cell wall arabinogalactan is one important basic approach for treatment of mycobacterial diseases. However, the biosynthetic origin of the D-arabinosyl monosaccharide residues themselves is not known. To obtain information on this issue, mycobacteria growing in culture were fed glucose labeled with 14 C or 3 H in specific positions. The resulting radiolabeled cell walls were isolated and hydrolyzed, the arabinose and galactose were separated by high-pressure liquid chromatography, and the radioactivity in each sugar was determined. [U- H]glucose were shown to be C-1 and H-5, respectively. These results demonstrated that the arabinose carbon skeleton is formed via the nonoxidative pentose shunt and not via hexose decarboxylation or via triose condensations. Since the pentose shunt product, ribulose-5-phosphate, is converted to arabinose-5-phosphate as the first step in 3-keto-D-manno-octulosonic acid biosynthesis by gram-negative bacteria, such a conversion was then searched for in mycobacteria. However, cell-free enzymatic analysis using both phosphorous nuclear magnetic resonance spectrometry and colorimetric methods failed to detect the conversion. Thus, the conversion of the pentose shunt intermediates to the D-arabino stereochemistry is not via the expected isomerase but rather must occur via novel metabolic transformations.The structure (Fig. 1) of the mycobacterial cell wall arabinan moiety of the arabinogalactan (henceforth referred to simply as arabinan) reveals that it is a very complex branched polysaccharide of arabinofuranosyl (Araf) resides (1). It provides a key structural connection between the peptidoglycan and the mycolic acids (9). Thus, the mycolic acids are attached in a specific fashion to a nonreducing end pentaarabinofuranosideThe reducing end of the arabinan is attached to a galactofuran (1) which is finally attached to the peptidoglycan via the actinomycete-specific linker disaccharide [34-Rha-(133)-GlcNAc-(13phosphate)] (10). This structural information suggests that arabinan is essential for cell wall integrity, and the antimycobacterial activity of ethambutol, an inhibitor of arabinan formation (3, 14), confirms this expectation.In parallel with what is known about the biosynthesis of other polysaccharides (12), it is expected that the arabinan is synthesized via arabinosyltransferases. These enzymes should utilize a 1-phosphorylated arabinose (either an arabinosyl sugar nucleotide or an arabinosyl polyprenyl phosphate) as the donor and the incomplete growing arabinan as the acceptor. Indeed, ␤-D-Araf-monophosphodecaprenol has been isolated and characterized from a variety of mycobacteria (18,19), and it has been demonstrated to function as an arabinosyl donor (7a, 21). It remains unknown if ␤-D-Araf-monophosphodecaprenol is the sole donor of arabinosyl residues or if there is an aqueous soluble arabinosyl nucleotide donor as well. In this regard, a preliminary report of a partially purifie...

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

“…All reproducible results now suggest that no sugar nucleotide of D-arabinofuranose is present in mycobacteria. The initial clue as to the precursors of D-arabinose came from feeding hexoses labeled in specific carbons to mycobacteria (9), which strongly suggested that the D-arabinose five-carbon skeleton was formed in the pentose shunt. A major step forward (10) then followed when it was recognized that membrane fractions of M. smegmatis were capable of converting pRpp to decaprenylphosphoryl-D-arabinose and, furthermore, that DPPR was an intermediate.…”

“…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).…”

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

“…The D-arabinofuran residues (Araf) and D-galactofuran residues (Galf) are added to form the mature lipid-linked AG, and the immediate donor of the polymerized Araf is decaprenylphosphoryl-D-arabinose (DPA) contributing to AG and lipoarabinomannan (LAM) assembly [3][4][5]. Many of the enzymes catalyzing the synthesis of DPA serving as an arabinose donor in the cell wall biosynthesis have been identified [6] and utilized as the targets for the development of new drugs against TB [7,8].…”

Metabolism of Plasma Membrane Lipids in Mycobacteria and Corynebacteria

“…The Araf portion is derived from the pentose-phosphate pathway [159][160][161]. 5-phosphoribose 1-diphosphate is transferred to Dec-P by the Rv3806c gene product [162] and the resultant Dec-P--D-5-phosphoribose is dephosphorylated to form Dec-P--D-ribose.…”