Recombinant Mycobacterium tuberculosis KatG(S315T) is a competent catalase-peroxidase with reduced activity toward isoniazid.
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.
Wengenack
N L
NL
Department of Biochemistry and Molecular Biology, and Biomedical Mass Spectrometry Facility, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Uhl
J R
JR
St Amand
A L
AL
Tomlinson
A J
AJ
Benson
L M
LM
Naylor
S
S
Kline
B C
BC
Cockerill
F R
FR
Rusnak
F
F
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
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010544:Peroxidases; D002374:Catalase; C076136:catalase HPI; D007538:Isoniazid