The crystal structure of 1-D-myo-inosityl-2-acetamido-2-deoxy-α-D-glucopyranoside deacetylase (MshB) from Mycobacterium tuberculosis reveals a zinc hydrolase with a lactate dehydrogenase fold

Mycothiol ( 1- D- myo- inosityl 2-( N- acetyl- L- cysteinyl)amido- 2- deoxy- alpha- D- glucopyranoside, MSH or AcCys-GlcN- inositol ( Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc- Ins to yield GlcN- Ins. The deacetylase ( MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn2+ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x- ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N- terminal domain and a C-terminal domain consisting of two beta- sheets and two alpha- helices. The zinc binding site is in the N- terminal domain occupying a position equivalent to that of the NAD(+) co- factor of lactate dehydrogenase. The Zn2+ is 5 coordinate with 3 residues from MshB ( His- 13, Asp- 16, His- 147) and two water molecules. One water would be displaced upon binding of substrate ( GlcNAc- Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp- 15. In addition to the Zn2+ providing electrophilic assistance in the hydrolysis, His- 144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S- conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB.