Characterization of BshA, bacillithiol glycosyltransferase from Staphylococcus aureus and Bacillus subtilis

Abstract

The first step during bacillithiol (BSH) biosynthesis involves the formation of N-acetylglucosaminylmalate from UDP-N-acetylglucosamine and l-malate and is catalyzed by a GT4 class glycosyltransferase enzyme (BshA). Recombinant Staphylococcus aureus and Bacillus subtilis BshA were highly specific and active with l-malate but the former showed low activity with d-glyceric acid and the latter with d-malate. We show that BshA is inhibited by BSH and similarly that MshA (first enzyme of mycothiol biosynthesis) is inhibited by the final product MSH.

Fig. 1

Structure of a) mycothiol, b) bacillithiol, c) O-UDP-N-acetylglucosamine (oxidized UDP-N-acetylglucosamine); note that the ribose moiety in UDP-N-acetylglucosamine has been oxidized to a dialdehydo- moiety, and d) BshA glycosyl transferase reaction

Fig. 2

3D structure of B. subtilis and S. aureus BshA, a) B. anthracis BshA (BA1558), b) B. subtilis BshA, and c) S. aureus BshA. Residues identified in S. aureus and B. subtilis are equivalent to B. anthracis residue numbers.

Fig. 3

Inhibiton of B. subtilis BshA by BSH. a) B. subtilis BshA (0.71 μg) preincubated with BSH and assayed for BshA activity with 0.5mM UDP-GlcNAc and 0.5 mM L-malate

Fig. 4

a) M. smegmatis cell free extract (2.7 mg total protein) preincubated with MSH and assayed for MshA activity with 1mM UDP-GlcNAc and 1mM 1-L-inositiol-1-phosphate. b) M. smegmatis cell free extract preincubated with O-UDP-GlcNAc and assayed for MshA activity with 1 mM UDP-GlcNAc and 1 mM 1-L-inositiol-1-phosphate. Control MshA rates, without inhibitor, were 0.4 nmoles min^-1 mg protein^-1.