title: Purification and characterization of the Mycobacterium smegmatis catalase-peroxidase involved in isoniazid activation.
abstract: The unique antitubercular activity of isoniazid requires that the drug be oxidized by the katG-encoded mycobacterial catalase-peroxidase to an activated drug form. In order to quantitatively assess the catalytic capabilities of the enzyme, the native catalase-peroxidase from Mycobacterium smegmatis was purified over 200-fold to homogeneity. The enzyme was shown to exhibit both catalase and peroxidase activities, and in the presence of either hydrogen peroxide or t-butyl peroxide, was found to catalyze the oxidation of the reduced pyridine nucleotides, NADH and NADPH, as well as artificial peroxidase substrates, at rates between 2.7 and 20 s-1. The homogeneous enzyme exhibited a visible absorbance spectrum typical of ferric heme-containing catalase-peroxidases, with a Soret maximum at 406 nm. Low temperature (10 K) electron paramagnetic resonance spectra in the presence of ethylene glycol revealed a high spin Fe(III) signal with g values of 5.9 and 5.6. The enzyme was very slowly (t1/2 = approximately 20 min) reduced by dithionite, and the reduced form showed typical spectral changes when either KCN or CO were subsequently added. The M. smegmatis catalase-peroxidase was found to contain 2 heme molecules per tetramer, which were identified as iron protoporphyrin IX by the pyridine hemochromogen assay. The peroxidatic activity was inhibited by KCN, NaN3, isoniazid (isonicotinic acid hydrazide), and its isomer, nicotinic acid hydrazide, but not by 3-amino-1,2,4-triazole. The role of mycobacterial catalase-peroxidases in the oxidative activation of the antitubercular prodrug isoniazid is discussed.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Marcinkeviciene
authors.forename: R S
authors.initials: RS
authors.lastname: Magliozzo
authors.forename: J S
authors.initials: JS
authors.lastname: Blanchard
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D002374:Catalase / Q000302:isolation & purification* / Q000378:metabolism; D003962:Dianisidine / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D007538:Isoniazid / Q000378:metabolism*; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000201:enzymology*; D010544:Peroxidases / Q000302:isolation & purification* / Q000378:metabolism; D011355:Prodrugs / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis
chemical_list: D001426:Bacterial Proteins; D011355:Prodrugs; D010544:Peroxidases; D002374:Catalase; C076136:catalase HPI; D003962:Dianisidine; D007538:Isoniazid