Abstract
Catalase-peroxidase (KatG) from Mycobacterium tuberculosis, a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid-based radicals during turnover with alkyl peroxides, and this work focuses on extending the identification and characterization of radicals forming on the millisecond to second time scale. Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme pocket binding of the drug and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Tyr(229). High field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal, and new analyses of X-band RFQ-EPR spectra also established its presence. High field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on x-ray structural data for particular tyrosine and tryptophan residues, enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F, and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Tyr(229) within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species and finally to only a tyrosyl radical on residue Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena.
Highlights
Mycobacterium tuberculosis catalase-peroxidase (KatG)3 is the enzyme responsible for activation of the anti-TB drug isoniazid (INH) in use for over fifty years, and mutations in this enzyme are the primary source of drug resistance in clinical strains of the TB pathogen throughout the world [1,2,3]
We suggested in a previous report that radicals could have a catalytic function, because they were quenched in reactions with INH [10]
Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) Spectroscopy of Ferric KatG Plus Peroxide and INH— The RFQ-EPR approach used here has been described previously; briefly, resting enzyme is mixed with a 3-fold molar excess of alkyl peroxide, and sample mixtures are frozen in liquid isopentane at Ϫ130 °C in precision bore EPR tubes
Summary
KatG, catalase-peroxidase; Mtb, M. tuberculosis; WT, wild type; KatG[W107F] and KatG[W91F], W107F and W91F mutants of KatG, respectively; INH, isonicotinic acid hydrazide; PAA, peroxyacetic acid; EPR, electron paramagnetic resonance; RFQ-EPR, rapid freeze-quench EPR; HF-EPR, high field EPR; TB, tuberculosis; WD, wide doublet; ND, narrow doublet; WS, wide singlet; NS, narrow singlet. Trapping and other techniques have been applied to gain insights into radical formation and identity in a collection of metalloenzymes Both these approaches have allowed identification of tyrosyl and tryptophanyl radicals in KatG enzymes and some assignment of the residues on which they reside [12, 13]. Evidence points to a unique catalytic requirement in catalase-peroxidases of three amino acids on the distal side of the heme, which appear in a covalent adduct connecting the side chains of Trp107, Tyr229, and Met255 in all KatG crystal structures (Fig. 1) [6, 7, 14]. The results overall are consistent with radicals formed initially on residues Tyr229 and Trp107 closest to the heme, followed by the appearance of additional radical(s) at more distant sites
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