title: Recombinant Mycobacterium tuberculosis KatG(S315T) is a competent catalase-peroxidase with reduced activity toward isoniazid.
abstract: The presence of KatG(S315T), a mutation frequently detected in clinical isolates of Mycobacterium tuberculosis, has been associated with loss of catalase-peroxidase activity and resistance to isoniazid therapy. Wild-type KatG and KatG(S315T) were expressed in a heterologous host (Escherichia coli) and purified to homogeneity, and enzymatic activity was measured. The catalase activity for KatG(S315T) was reduced 6-fold, and its peroxidase activity was decreased <2-fold, compared with the activities for wild-type KatG. Pyridine hemochrome analysis demonstrated 1.1 +/- 0.1 hemes/subunit for wild-type KatG and 0.9 +/- 0.1 hemes/subunit for KatG(S315T), indicating that the difference in enzymatic activity is not the result of incomplete heme cofactor incorporation in KatG(S315T). High-performance liquid chromatography analysis showed that wild-type KatG was more efficient than KatG(S315T) at converting isoniazid to isonicotinic acid. These results demonstrate that KatG(S315T), as expressed in E. coli, is a competent catalase-peroxidase that exhibits a reduced ability to metabolize isoniazid.
authors.affiliation: Department of Biochemistry and Molecular Biology, and Biomedical Mass Spectrometry Facility, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
authors.forename: N L
authors.initials: NL
authors.lastname: Wengenack
authors.forename: J R
authors.initials: JR
authors.lastname: Uhl
authors.forename: A L
authors.initials: AL
authors.lastname: St Amand
authors.forename: A J
authors.initials: AJ
authors.lastname: Tomlinson
authors.forename: L M
authors.initials: LM
authors.lastname: Benson
authors.forename: S
authors.initials: S
authors.lastname: Naylor
authors.forename: B C
authors.initials: BC
authors.lastname: Kline
authors.forename: F R
authors.initials: FR
authors.lastname: Cockerill
authors.forename: F
authors.initials: F
authors.lastname: Rusnak
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002374:Catalase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli; D007538:Isoniazid / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000201:enzymology*; D010544:Peroxidases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010544:Peroxidases; D002374:Catalase; C076136:catalase HPI; D007538:Isoniazid