Mycothiol (
MSH) is the predominant low molecular weight
thiol produced by actinomycetes, and it plays a pivotal role in the bacterial detoxication process. 1L-myo-Inositol-1-phosphate (1L-Ins-1-P) α-
N-acetylglucosaminyltransferase (GlcNAc-T), known as MshA, is the only
glycosyltransferase involved in
MSH biosynthesis. In this work, the MshA from Corynebacterium
diphtheria, named as CdMshA, was expressed, purified, and studied in detail. Its enzymatic activity to transfer GlcNAc to 1L-Ins-1-P was confirmed by the isolation and rigorous characterization of its reaction product 3-phospho-1-d-myo-inositol-2-acetamido-2-deoxy-α-d-glucopyranoside. CdMshA was shown to accept only
UDP-GlcNAc and 1L-Ins-1-P as its substrates among various tested glycosyl donors, such as
UDP-GlcNAc,
UDP-Gal,
UDP-Glc,
UDP-GalNAc and
UDP-GlcA, and glycosyl acceptors, such as myo-
inositol, 1L-Ins-1-P and 1D-Ins-1-P. The results have demonstrated the strict substrate selectivity of CdMshA. Furthermore, its reaction kinetics with
UDP-GlcNAc and 1L-Ins-1-P as substrates were characterized, while site-directed mutagenesis of CdMshA disclosed that its
amino acid residues N28, K81 and R157 were essential for its enzymatic activity.