Mycothiol (MSH), a low-molecular- weight thiol, is a primary reducing agent and essential for the survival of mycobacteria. The full pathway of MSH biosynthesis and detoxification includes various promising drug targets. Several metalloenzymes are involved in this pathway, such as a deacetylase (MshB) and mycothiol S-conjugate amidase (MCA). MshB catalyzes the deacetylation of GlcNAc-Ins to form GlcN-Ins and acetate. Mycothiol S-conjugate amidase (MCA) cleaves the amide bond of mycothiol S-conjugates of various drugs and toxins. The identification of the native co-factor is critical for the design of potent and effective inhibitors. Therefore, in this study, we identified the possible native co-factors of MshB and MCA from M. smegmatis and M. tuberculosis. To reach our aim, we used a pull-down method to rapidly purify halo-MshB and halo-MCA under anaerobic conditions. Our data indicates that the metal bound to MshB and MCA anaerobically purified from E. coli grown in minimal medium is mainly Fe(II), while proteins purified under aerobic conditions contain bound Zn (II) and Fe(II) that varies with the metal content of the medium. For a further clarification of the metal ion preferences of MshB and MCA, we determined the MshB and MCA affinity for Zn(II) to be in the picomolar range and Ms MshB affinity for Fe(II) in nanomolar range. These results indicate that MshB and MCA can be found bound with either iron or zinc and this is independent to their affinities for these metal ions. / Master of Science

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. / Ph. D.