Enzymatic Characterization of N-Acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside Deacetylase (MshB)
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Mycobacterium species, which contain the causative agent for human tuberculosis (TB), produce inositol derivatives including mycothiol (MSH). MSH is a unique and dominant cytosolic thiol that protects mycobacterial pathogens against the damaging effects of reactive oxygen species and is involved in antibiotic detoxification. Therefore, MSH is considered a potential drug target. The deacetylase MshB catalyzes the committed step in MSH biosynthesis by converting N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins) to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins). In this dissertation, we present detailed functional analysis of MshB. Our work has shown that MshB is activated by divalent metal ions that can switch between Zn2+ and Fe2+ depending on environmental conditions, including metal ion availability and oxidative conditions. MshB employs a general acid-base catalyst mechanism wherein the Asp15 functions as a general base to activate the metal-bound water nucleophile for attack of the carbonyl carbon on substrate. Proton-transfer from a general acid catalyst facilitates breakdown of the tetrahedral intermediate and release of products. A dynamic tyrosine was identified that regulates access to the active site and participates in catalysis by stabilizing the oxyanion intermediate. Molecular docking simulations suggest that the GlcNAc moiety on GlcNAc-Ins is stabilized by hydrogen bonding interactions with active site residues, while a hydrophobic stacking interaction between the inositol ring and Met98 also appears to contribute to substrate affinity for MshB. Additional binding interactions with side chains in a hydrophobic cavity adjacent to the active site were suggested when the docking experiments were carried out with large amidase substrates. Together the results from this study provide groundwork for the rational design of specific inhibitors against MshB, which may circumvent current challenges with TB treatment.
- Doctoral Dissertations