Radical Sites in Mycobacterium tuberculosis KatG Identified Using Electron Paramagnetic Resonance Spectroscopy, the Three-dimensional Crystal Structure, and Electron Transfer Couplings

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.

Antibiotic Resistance in Mycobacterium tuberculosis

“…S2). Furthermore, the RFQ-EPR results did not show an increase in the yield of protein-based radicals, such as the tyrosyl and tryptophanyl radicals assigned in WT KatG (40,41), which are formed under similar conditions in the presence of PAA. One additional finding was that anaerobic preparation of RFQ-EPR samples of enzyme treated with PAA did not alter the identity of the signals observed to any great extent (not shown) compared with aerobic samples, arguing against a peroxyl radical produced from reaction of dioxygen with an amino acid radical.…”

“…ϩ intermediate is also of interest, since this process is known to involve endogenous electron transfers producing radicals on amino acids (40,41,48) and could have an impact on INH activation. Interestingly, the time course for this return was ϳ2 times faster in the mutant than in WT KatG (Fig.…”

“…In the absence of reducing substrates, the reaction of WT KatG with excess alkyl peroxides generates tyrosyl and tryptophanyl radicals (40,41), which contributes to the quenching of heme intermediates. However, no direct evidence for an increased yield of amino acid-based radicals was found in RFQ-EPR experiments with KatG[S315G]; the maximum total yield of radical in the case of the mutant was in fact lower by 60% during the first 200 ms of the reaction, compared with the yield in WT KatG.…”

An Oxyferrous Heme/Protein-based Radical Intermediate Is Catalytically Competent in the Catalase Reaction of Mycobacterium tuberculosis Catalase-Peroxidase (KatG)

“…The presence of the mutated codons in the katG gene was confirmed by DNA sequencing (Gene Wiz, Inc.), and the mutated plasmid was electroporated into E. coli strain UM262 for protein overexpression. The mutants Y229F and W107F were prepared as reported previously (7,22).…”

Role of the Oxyferrous Heme Intermediate and Distal Side Adduct Radical in the Catalase Activity of Mycobacterium tuberculosis KatG Revealed by the W107F Mutant

“…Construction, Expression, and Purification of Distal MutantsOverexpression of recombinant WT KatG and KatG mutant enzymes and their purification were achieved as described in the companion paper (52) and previous reports (14,21).…”