Examination of mechanism of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) reveals unexpected role for dynamic tyrosine.

Actinomycetes are a group of gram-positive bacteria that includes pathogenic mycobacterial species, such as Mycobacterium tuberculosis. These organisms do not have glutathione and instead utilize the small molecule mycothiol (MSH) as their primary reducing agent and for the detoxification of xenobiotics. Due to these important functions, enzymes involved in MSH biosynthesis and MSH-dependent detoxification are targets for drug development. The metal-dependent deacetylase N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) catalyzes the hydrolysis of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside to form 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside and acetate in MSH biosynthesis. Herein we examine the chemical mechanism of MshB. We demonstrate that the side chains of Asp-15, Tyr-142, His-144, and Asp-146 are important for catalytic activity. We show that NaF is an uncompetitive inhibitor of MshB, consistent with a metal-water/hydroxide functioning as the reactive nucleophile in the catalytic mechanism. We have previously shown that MshB activity has a bell-shaped dependence on pH with pK(a) values of ∼7.3 and 10.5 (Huang, X., Kocabas, E. and Hernick, M. (2011) J. Biol. Chem. 2...Continue Reading