Exploring the Substrate Specificity of a Mycobacterial Polyprenol Monophosphomannose‐Dependent α‐(1→6)‐Mannosyltransferase

Abstract

A series of synthetic alpha-(1-->6)-linked octyl mannopyranoside oligomers was evaluated as potential acceptors of a polyprenol monophosphomannose-dependent alpha-(1-->6)-mannosyltransferase that is involved in the biosynthesis of the mannan core of mycobacterial lipoarabinomannan. Initial evaluation demonstrated that the enzyme recognizes di-, tri- and tetramannosides (5, 6 and 7) as substrates with different activities. While the highest mannosyltransferase activities were observed when the di- and trisaccharide were used as substrates, diminished enzymatic activity was seen with the tetramannoside. As octyl alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranoside (5) appears to be the minimum structural element required for mannosyltransferase catalysis, a panel of methoxy and deoxy disaccharide analogues (8-21) were used to probe the substrate specificity of the enzyme further. In terms of the steric requirements at the active site, the enzyme does not recognize either C2'- and C2-methoxy analogues as substrates, a result that suggests that the alpha-(1-->2)-mannopyranosyl branches, which are present in the mannan core of LAM must be added on a larger alpha-(1-->6)-oligomannan intermediate. In contrast, the presence of a methoxy functionality at the C3', C3, C4' and C4 positions are somewhat tolerated by the enzyme, although diminished enzyme activities were observed with the C4'- and C4-methoxy analogues. Moreover, the 2'- and 4-hydroxyl groups appear not to be critical for substrate binding at the active site, as both 2'- and 4-deoxy analogues are substrates for the enzyme. In contrast, replacement of the hydroxyl groups at other positions essentially abolished enzymatic activity. Further kinetic characterization of the enzyme by using the effective acceptor substrates gave apparent K(M) values ranging from 111 to 437 microM, which are within two-fold higher or lower than that for the parent dimannoside (5). Although the K(M) values indicate that the enzyme binds those acceptors with comparable affinities, the C4'-methoxy analogue (12) turns over more slowly than the others, as indicated by the apparent V(max) values.

Antitubercular Drugs Based on Carbohydrate Derivatives

“…The α-(1→6)-mannopyranose Manp (1→6)-α-Manp motif found in the mannan core ( Fig. 18.13.1) of the LAM was identified as minimum acceptor motif for enzymes synthesizing the LAM mannan structure [187,188,193,200]. These studies also revealed substrate specificity, optimal length, and key interactions with the α-(1,6)-ManT involved in the LAM biosynthesis.…”

“…Efforts to develop inhibitors and substrates for α-(1,6)-ManT involved in LM and LAM biosynthesis have also been reported [187][188][189][190][191][192][193][194]. For example, a triazole-linked α-1,6-C-oligomannoside up to hexadecamer is reported [192].…”

“…There have been notable contributions in developing α-(1,6)-ManT inhibitors and substrates [187,188,193]. The α-(1→6)-mannopyranose Manp (1→6)-α-Manp motif found in the mannan core ( Fig.…”

“…A 6-deoxyamino analog was a weak inhibitor and showed 50% inhibition of activity at ~1 mM concentration [193]. Other analogs of the Manp-(1→6)-α-Manp Octyl glycosides, such as deoxy, methoxy, fluoro, and amino functionalities were also synthesized and evaluated as substrates and inhibitors of PPMdependent α-(1→6)-ManT [188,190,191,194].…”

“…Mannan core found in the LAM complex,[39,48] 2. α-Manp-(1→6)-α-Manp Octyl glycoside (minimum acceptor)[187,188], 3. and 4. D-galactose-phosphonate analogs of myo-inositol-1-phosphate and PI.…”