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

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.