Revisiting the Specificity of an α‐(1→4)‐Mannosyltransferase Involved in Mycobacterial Methylmannose Polysaccharide Biosynthesis

Abstract

In order to evaluate the proposed biosynthetic pathway for the methylmannose (MMPs) polysaccharides produced by mycobacteria, two homologous series of synthetic α-(1→4)-linked 3-O-methyl-mannopyranosides, one terminated at the non-reducing end by a free mannopyranose residue (unmethylated oligosaccharides; OS) and the other terminated by a 3-O-methyl-mannopyranose residue (methylated OS), were prepared and evaluated as potential acceptors of an α-(1→4)-mannosyltransferase. Using a mycobacterial membrane preparation as the source of the transferase, it was found that unmethylated OS are better substrates for the enzyme compared to the methylated OS of the same length. These results are inconsistent with the proposed MMP biosynthetic pathway, which suggests only methylated OS are acceptors of this transferase. To confirm that the observed activity arose from the desired α-(1→4)-mannosyltransferase, as opposed to other mannosyltransferases present in the membrane preparation, the products resulting from tetrasaccharides 4 (unmethylated OS) and 9 (methylated OS), which only differ in the terminal residue, were further analyzed. MALDI-MS, exo-glycosidase digestion and (1) H NMR spectroscopy were used to evaluate the structures of these reaction products. These experiments revealed that the enzymatic products of both 4 and 9 contain only α-(1→4)-linked mannose residues, confirming the activity of the α-(1→4)-mannosyltransferase. This supports the finding that both methylated and unmethylated OS are acceptors of the enzyme. It was also demonstrated that a homologous series of oligosaccharides with different number of mannose residues were formed from both 4 and 9, as opposed to a single reaction product. These results, again, challenge the previously proposed MMP biosynthetic pathway involving alternating methylation and mannosylation reactions.

Studies on the substrate specificity of a GDP-mannose pyrophosphorylase from Salmonella enterica

“…As part of a larger study on the specificity of mannosyltransferases involved in mycobacterial glycan biosynthesis [20–22], we had the need for a panel of singly deoxygenated and methylated guanosine diphosphosphate mannopyranose (GDP-Man) derivatives. In developing a strategy for the synthesis of these compounds, we chose to take advantage of a GDP-mannose pyrophosphorylase (GDP-ManPP) from Salmonella enterica [23], which had previously been shown to have a relaxed specificity for the sugar 1-phosphate moiety [24–25].…”

Antitubercular Drugs Based on Carbohydrate Derivatives

“…Two series of α-(1→4)-linked 3-O-methyl-mannopyranosides with either 3-O-methyl or free mannopyranose terminating residues were synthesized and tested as acceptors of the α-(1→4)-ManT [186]. The result suggests that ManT could produce linear MMPs independent of OMT and contradicts the joint activity between ManT and OMT [186].…”

“…Proposed MMP biosynthesis follows elongation with alternate mannosylation via α-(1→4)-ManT, and methylation via 3-O-methyltransferase (OMT) [183,185]. Recently, an investigation on specificity of the ManT involved in MMPs biosynthesis was reported [186]. Two series of α-(1→4)-linked 3-O-methyl-mannopyranosides with either 3-O-methyl or free mannopyranose terminating residues were synthesized and tested as acceptors of the α-(1→4)-ManT [186].…”

Amphiphilic Cytosolic Glycans from Mycobacteria: Occurrence, Lipid‐Binding Properties, Biosynthesis, and Synthesis

“…To probe this biosynthetic pathway further, the substrate specificity of ManT was recently studied using synthetic substrate analogs of ManT and OMT . In this study, it was demonstrated that ManT indeed recognizes synthetic ManT precursors that have a methyl group at the non‐reducing end.…”