Biosynthesis of mycothiol: elucidation of the sequence of steps in Mycobacterium smegmatis

Abstract

Several members of the Actinomycetales, including the medically important mycobacteria, produce 1-D-myo-inosityl-2-(N-acetyl-l-cysteinyl)amino-2-deoxy-α-d-glucopyranoside (trivial name mycothiol) as their principal low-molecular-mass thiol. The pseudo-disaccharide component of mycothiol, 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (α-D-GI), was synthesized by ligation of 1-D,l-2,3,4,5,6-penta-O-acetyl-myo-inositol to 3,4,6-tri-O-acetyl-2-deoxy-2-(2,4-dinitrophenylamino)-α-D-glu-copyranosyl bromide to give, in the first instance, an isomeric mixture of α- and β-linked pseudo-disaccharides. The α-coupled d,D and D,l isomers, α-d-GI and α-l-GI respectively, were purified from the mixture by TLC, followed by removal of the protecting groups. A cell-free extract of Mycobacterium smegmatis catalysed the ligation of cysteine, acetate and α-D-GI in the presence of ATP and Mg2+ to form mycothiol, as judged by HPLC. When no acetate was added to the incubation mixture, an additional thiol accumulated. In the presence of [14C]acetate no radiolabel was recovered in this species, but only in mycothiol. The additional thiol was isolated as the bimane derivative, and 1H and 1H–1H COSY NMR spectra confirmed its identity as desacetylmycothiol. A more complete conversion of desacetylmycothiol into mycothiol was achieved in the presence of acetyl-S-CoA. These results indicate that the biosynthesis of mycothiol proceeds by the sequential addition of cysteine and acetate to α-D-GI. The inositol moiety appears to be an important determinant of specificity, since α-L-GI was poorly utilized.