Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts

6-POS Board 6 Crystallization and Structural Characterization of Mycothiol S-Conjugate Amidase from Mycobacterium Tuberculosis Jeremy G. Burgess , Bryan T. Sewell, Brandon W. Weber. University of Cape Town, Cape Town, South Africa. Mycothiol (MSH) is a protective thiol found within Mycobaterium tuberculosis and other Actinomycetes. MSH is an intracellular protectant, used to counteract oxidative stress and to detoxify endogenous toxins and xenobiotics. Accordingly, enzymes needed for MSH biosynthesis and metabolism have received interest due to their potential as novel drug targets. Mycothiol S-conjugate amidase (Mca), is a zinc metalloenzyme and member of the LmbE-like superfamily, which catalyzes the hydrolysis of mycothiol S-conjugates (MSR) to glucosaminyl- inositol (GlcN-Ins) and an acetylcysteine S-conjugate (AcCyS-R), which is expelled from the cell. Mca shares minor overlapping substrate specificity with MshB, a deacetylase involved in MSH biosynthesis and a fellow member of the LmbE-like superfamily. Several unconserved residues in the GlcN-Ins binding site of Mca and MshB are thought to be responsible for the observed differences in substrate specificity for Mca and MshB, but their precise contributions are unknown. Site directed mutagenesis and activity assays will be used to analyze the effect of substitution of these residues in Mca with their MshB counterparts. Native Mca was previously produced in E.coli as a fusion protein with maltose binding protein (MalE). The fusion protein was purified by affinity chromatography and cleaved using Tobacco Etch Virus protease. Free Mca activity was demonstrated through a discontinuous assay. Early sparse-matrix crystallization screens with native Mca were unsuccessful. Stabilizing additives identified through thermostability assays were included in later crystallization screens, but the conditions require optimization. Additional attempts to crystallize Mca will include (a) generation of inactive Mca mutants and incubation with a favoured substrate, and (b) removal of disordered regions within Mca, including the final twenty C-terminal residues.

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