Overexpression, purification, and characterization of the catalase-peroxidase KatG from Mycobacterium tuberculosis.
Wild-type catalase-peroxidase KatG from Mycobacterium tuberculosis as well as a specific mutant (R463L) frequently found in isoniazid-resistant strains have been overexpressed in Escherichia coli, allowing purification of sufficient quantities of enzyme for physical and kinetic characterization. Optical absorption and EPR spectroscopies indicate that KatG is similar to a growing class of bacterial catalase-peroxidases. Optical and EPR spectra of KatG in the presence of either a strong field or weak field ligand suggest that, like horseradish peroxidase and metmyoglobin, KatG is likely to have a histidine as a proximal ligand. The wild-type enzyme functions as a highly active catalase as well as a broad specificity peroxidase. Wild-type KatG and the R463L mutant of KatG exhibit identical spectroscopic and kinetic properties. Furthermore, both enzymes are equally capable of metabolizing the important antituberculosis drug isoniazid.
Johnsson
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K
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California 94720, USA.
Froland
W A
WA
Schultz
P G
PG
D001426:Bacterial Proteins; D002374:Catalase / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007538:Isoniazid / Q000378:metabolism; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D010544:Peroxidases; D002374:Catalase; C076136:catalase HPI; D007538:Isoniazid