Purification and characterization of Mycobacterium tuberculosis KatG, KatG(S315T), and Mycobacterium bovis KatG(R463L)

Protein Expression and Purification
N L WengenackF Rusnak

Abstract

Isoniazid, a first-line antibiotic used for the treatment of tuberculosis, is a prodrug that requires activation by the Mycobacterium tuberculosis enzyme KatG. The KatG(S315T) mutation causes isoniazid resistance while the KatG(R463L) variation is thought to be a polymorphism. Much of the work to date focused on isoniazid activation by KatG has utilized recombinant enzyme overexpressed in Escherichia coli. In this work, native KatG and KatG(S315T) were purified from M. tuberculosis, and KatG(R463L) was purified from Mycobacterium bovis. The native molecular weight, enzymatic activity, optical, resonance Raman, and EPR spectra, K(D) for isoniazid binding, and isoniazid oxidation rates were measured and compared for each native enzyme. Further, the properties of the native enzymes were compared and contrasted with those reported for recombinant KatG, KatG(S315T), and KatG(R463L) in order to assess the ability of the recombinant enzymes to act as good models for the native enzymes.

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Citations

Nov 28, 2013·Archives of Biochemistry and Biophysics·Olive J NjumaDouglas C Goodwin
Oct 15, 2011·Bioresource Technology·Shefali SangarRavinder S Jolly
Jan 8, 2010·Protein Science : a Publication of the Protein Society·Christine E CadeReza A Ghiladi
May 16, 2015·The Journal of Antimicrobial Chemotherapy·Elena MartinezVitali Sintchenko
Apr 4, 2017·Molecular Biology and Evolution·Anastasia S KochRobert J Wilkinson
Oct 26, 2005·The Journal of Biological Chemistry·Christa JakopitschChristian Obinger

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