N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) is a key enzyme in mycothiol biosynthesis

Mycothiol is a novel thiol produced only by actinomycetes and is the major low-molecular-weight thiol in mycobacteria, Mycothiol was previously shown to be synthesized from 1-D-myo-inosityl-2-amino-2-deoxy-alpha -D-glucopyran oside by ligation with cysteine followed by acetylation, A novel mycothiol- dependent detoxification enzyme, mycothiol conjugate amidase, was recently identified in Mycobacterium smegmatis and shown to have a homolog, Rv1082, in Mycobacterium tuberculosis, In the present study we found that a protein encoded by the M, tuberculosis open reading frame Rv1170, a homolog of Rv1 082, possesses weak mycothiol conjugate amidase activity but shows substant ial deacetylation activity with 1-D-myo-inosityl-2-acetamido-2-deoxy-alpha -D-glucopyranoside (GlcNAc-Ins), a hypothetical mycothiol biosynthetic prec ursor. The availability of this protein enabled us to develop an assay for GlcNAc-Ins, which was used to demonstrate that GlcNAc-Ins is present in M, smegmatis at a level about twice that of mycothiol, It was shown that GlcNA c-Ins is absent in mycothiol-deficient mutant strain 49 of M, smegmatis and that this strain can concentrate GlcNAc-Ins from the medium and convert it to mycothiol, This demonstrates that GlcNAc-Ins is a key intermediate in t he pathway of mycothiol biosynthesis, Assignment of Rv1170 as the gene codi ng the deacetylase in the M, tuberculosis genome represents the first ident ification of a gene of the mycothiol biosynthesis pathway. The presence of a large cellular pool of substrate for this enzyme suggests that it may be important in regulating mycothiol biosynthesis.