Biochemistry. 2015 Jun 23;54(24):3784-90. doi: 10.1021/acs.biochem.5b00068.

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

 

Xinyi Huang1 and Marcy Hernick1,2*

1Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061; 2Department of Pharmaceutical Sciences, Appalachian College of Pharmacy, Oakwood, VA 24631

 

Abstract

Actinobacteria such as Mycobacterium tuberculosis use the unique thiol mycothiol (MSH) as their primary reducing agent and in the detoxification of xenobiotics. N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB) is the metal-dependent deacetylase that catalyzes the deacetylation of N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside, the committed step in MSH biosynthesis. We previously used docking studies to identify specific side chains that may contribute as molecular determinants of MshB substrate specificity [Huang, X., and Hernick, M. (2014) Biopolymers 101, 406-417]. Herein, we probe the molecular basis of N-acetylglucosamine (GlcNAc) recognition and turnover by MshB using a combination of site-directed mutagenesis and kinetic studies (mutants examined, L19A, E47A, R68A, D95A, M98A, D146N, and F216A). Results from these studies indicate that MshB is unable to catalyze the turnover of GlcNAc upon loss of the Arg68 or Asp95 side chains, consistent with the proposal that these side chains make critical hydrogen bonding interactions with substrate. The activity of the D146N mutant is ∼10-fold higher than that of the D146A mutant, suggesting that the ability to accept a hydrogen bond at this position contributes to GlcNAc substrate specificity. Because there does not appear to be a direct contact between Asp146 and substrate, this effect is likely mediated via positioning of other catalytically important residues. Finally, we probed side chains located on mobile loops and in a hydrophobic cavity and identified two additional side chains (Met98 and Glu47) that contribute to GlcNAc recognition and turnover by MshB. Together, results from these studies confirm some of the molecular determinants of GlcNAc substrate specificity by MshB, which should aid the development of MshB inhibitors.

PMID: 26024468

 

Additional Information

Tuberculosis (TB) is a disease that results from infection with the organism Mycobacterium tuberculosis. Although there are antibiotics available to treat TB, this disease continues to present a challenge and there were 1.5 million deaths worldwide attributed to TB in 2013.1 One of the difficulties in treating TB is the increasing presence of multidrug resistant (MDR)-TB with the number of patients diagnosed with MDR-TB tripling between 2009 and 2013.1

To tackle the problems associated with MDR-TB, new antibiotics are needed. Since antibiotic resistance is often linked with the drug target, drugs that act upon untapped targets are necessary. One potential source of targets in M. tuberculosis is the mycothiol biosynthetic and detoxification pathways.2,3 These pathways serve critical protective roles in the bacteria, shielding these organisms from our natural defense systems. Therefore, if we can discover a way to block the production of key metabolites in these pathways, we could restore the ability of our immune system to eradicate these organisms.

The work in this paper focuses on an enzyme (MshB) involved in an important step in the production of mycothiol. The goal of this project was to identify key interactions between MshB and ligands that could be exploited in the future for drug development. We previously used computational studies to predict potential enzyme-ligand interactions.4 In the current manuscript, we sought to confirm the predicted interactions using biochemical assays.

The side chains of seven amino acids that were predicted to make important interactions (e.g., ionic, hydrophobic, hydrogen bonding)4 were mutated to Ala and the effect on substrate turnover by MshB was measured using kinetic assays. Results from these studies confirm the importance of four side chains (Arg68, Asp95, Met98, Asp146), as mutation of these side chains significantly decreases the rate of substrate turnover by MshB indicating their importance for ligand recognition (Figure 1). Additionally, these studies have identified one side chain (Glu47) whose removal enhances turnover by MshB. We propose that this side chain is involved in regulating access to the enzyme active site.

The work described in this study has identified several key interactions that contribute to the recognition of ligands by MshB. Importantly, this work lays the foundation for the development of potent and specific MshB inhibitors that have the potential to serve as antibiotics for the treatment of TB.

 

 

MH FIG1

Figure 1. Model of substrate (N-acetylglucosamine) binding to MshB (PDB 4EWL).

 

References:

  1. WHO Global Tuberculosis Report, 2014.
  2. Hernick, M. Mycothiol, a target for potentiation of rifampin and other antibiotics against M. tuberculosis. Expert Review of Anti-Infective Therapy 2013, 11, 49-67
  3. Nilewar, S. S.; Kathiravan, M. K. Mycothiol: A promising antitubercular target. Bioorg. Chem. 2014, 52, 62-68
  4. Huang, X.; Hernick, M. 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). Biopolymers 2014, 101, 406-417

 

 

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