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Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Poster Abstracts
84
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.