Automated docking studies provide insights into molecular determinants of ligand recognition by N‐acetyl‐1‐d‐myo‐inosityl‐2‐amino‐2‐deoxy‐α‐d‐glucopyranoside deacetylase (MshB)

Abstract

The metal-dependent deacetylase N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) catalyzes the deacetylation of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcNAc-Ins), the committed step in mycothiol (MSH) biosynthesis. MSH is the thiol redox buffer used by mycobacteria to protect against oxidative damage and is involved in the detoxification of xenobiotics. As such, MshB is a target for the discovery of new drugs to treat tuberculosis (TB). While MshB substrate specificity and inhibitor activity have been probed extensively using enzyme kinetics, information regarding the molecular basis for the observed differences in substrate specificity and inhibitor activity is lacking. Herein we begin to examine the molecular determinants of MshB substrate specificity using automated docking studies with a set of known MshB substrates. Results from these studies offer insights into molecular recognition by MshB via identification of side chains and dynamic loops that may play roles in ligand binding. Additionally, results from these studies suggest that a hydrophobic cavity adjacent to the active site may be one important determinant of MshB substrate specificity. Importantly, this hydrophobic cavity may be advantageous for the design of MshB inhibitors with high affinity and specificity as potential TB drugs.

Molecular Determinants of N-Acetylglucosamine Recognition and Turnover by N-Acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside Deacetylase (MshB)

“…Docking studies with GlcNAc and MshB predicted this substrate is recognized via extensive hydrogen bonding interactions with the active site side chains of Arg68, Asp95, Tyr142, and His144 ( Figure 3). 12 We have previously confirmed the importance of Tyr142 and His144 in GlcNAc recognition and turnover by MshB, and here we report results from kinetic studies of the R68A and D95A mutants that confirm the importance of these side chains in GlcNAc recognition and turnover by MshB. Additionally, results from these kinetic studies also suggest a role for Met98 in GlcNAc recognition and turnover by MshB, while loss of the Glu47 side chain leads to an enhancement of GlcNAc turnover by MshB.…”

“…12 While Met98 cannot make a similar hydrophobic interaction with the smaller GlcNAc substrate, in one of the MshB-GlcNAc docking conformations the S atom on Met98 is 3.0 Å from the 1-OH of GlcNAc ( Figure 5A), suggesting it could accept a hydrogen bond from GlcNAc. To test this hypothesis, we examined the steady-state kinetics of the M98A mutant.…”

“…Specifically, the side chains of Arg68 and Asp95 both share hydrogen bonds with the bound glycerol in the Act-MshB structure 10 and bound BOG in the BOG-MshB 9 structure and are predicted to share hydrogen bonds with the glucosamine hydroxyl groups on GlcNAc (Figure 3). 12 While Arg68 is located on helix α2 and appears to be relatively rigid, Asp95 is located on a mobile surface loop (residues 95−104). Removal of either side chain with the R68A or D95A mutant results in an inactive MshB enzyme that has no measurable activity (Table 1) with the GlcNAc substrate [≤100 mM GlcNAc (R68A) or ≤300 mM GlcNAc (D95A)].…”

“…12,22 Because the side chains of Tyr142 (loop 2) and Phe216 (loop 3) are located in the proximity of one another in crystal structures of some of the MshB monomers ( Figure 5B), it is possible that a π−π interaction between Tyr142 and Phe216 could be coupled to movement of Tyr142. To test this hypothesis, we examined the steady-state kinetics of the F216A mutant.…”

“…11 We recently reported results from docking studies with a series of known MshB deacetylase and amidase substrates that identified a series of side chains that may play roles in ligand recognition and turnover by MshB ( Figure 3) that can be examined using biochemical and biophysical approaches. 12 These studies were conducted using Act-MshB as the receptor, because this is the only structure that captures the Tyr142 side chain in the position required to enhance the rate of chemistry.…”