Purification and characterization of Mycobacterium tuberculosis KatG, KatG(S315T), and Mycobacterium bovis KatG(R463L).
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.
Wengenack
Nancy L
NL
Department of Biochemistry and Molecular Biology and Section of Hematology Research, Mayo Clinic, Rochester, MN 55905, USA. wengenack.nancy@mayo.edu
Lane
Brian D
BD
Hill
Preston J
PJ
Uhl
James R
JR
Lukat-Rodgers
Gudrun S
GS
Hall
Leslie
L
Roberts
Glenn D
GD
Cockerill
Franklin R
FR
Brennan
Patrick J
PJ
Rodgers
Kenton R
KR
Belisle
John T
JT
Rusnak
Frank
F
D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002374:Catalase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D024881:Drug Resistance, Bacterial / Q000235:genetics; D007538:Isoniazid / Q000378:metabolism / Q000627:therapeutic use; D007700:Kinetics; D009163:Mycobacterium bovis / Q000201:enzymology* / Q000235:genetics; D010084:Oxidation-Reduction; D017354:Point Mutation / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis; D013379:Substrate Specificity / Q000235:genetics; D014376:Tuberculosis / Q000178:diet therapy / Q000382:microbiology / Q000473:pathology
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D007538:Isoniazid