BIOSYNTHESIS OF MYCOTHIOL - ELUCIDATION OF THE SEQUENCE OF STEPS IN MYCOBACTERIUM-SMEGMATIS

Several members of the Actinomycetales, including the medically import ant mycobacteria, produce -L-cysteinyl)amino-2-deoxy-alpha-D-glucopyra noside (trivial name mycothiol) as their principal low-molecular-mass thiol. The pseudo-disaccharide component of mycothiol, o-inosityl-2-am ino-2-deoxy-alpha-D-glucopyranoside (alpha-D-GI), was synthesized by l igation of 1-D,L-2,3,4,5,6-penta-O-acetyl-myo-inositol to -2-(2,4-dini trophenylamino)-alpha-D-glucopyranosyl bromide to give, in the first i nstance, an isomeric mixture of alpha- and beta-linked pseudo-disaccha rides. The alpha-coupled D,D and D,L isomers, alpha-D-GI and alpha-L-G I respectively, were purified from the mixture by TLC, followed by rem oval of the protecting groups. A cell-free extract of Mycobacterium sm egmatis catalysed the ligation of cysteine, acetate and alpha-D-GI in the presence of ATP and Mg2+ to form mycothiol, as judged by HPLC. Whe n no acetate was added to the incubation mixture, an additional thiol accumulated. In the presence of [C-14]acetate no radiolabel was recove red in this species, but only in mycothiol. The additional thiol was i solated as the bimane derivative, and H-1 and H-1-H-1 COSY NMR spectra confirmed its identity as desacetylmycothiol. A. more complete conver sion of desacetylmycothiol into mycothiol was achieved in the presence of acetyl-S-CoA. These results indicate that the biosynthesis of myco thiol proceeds by the sequential addition of cysteine and acetate to a lpha-D-GI. The inositol moiety appears to be an important determinant of specificity, since alpha-L-GI was poorly utilized.