Abstract
Catalase-peroxidases (KatG), which belong to Class I heme peroxidase enzymes, have high catalase activity and substantial peroxidase activity. The Y229F mutant of Mycobacterium tuberculosis KatG was prepared and characterized to investigate the functional role of this conserved residue unique to KatG enzymes. Purified, overexpressed KatG[Y229F] exhibited severely reduced steady-state catalase activity while the peroxidase activity was enhanced. Optical stopped-flow experiments showed rapid formation of Compound (Cmpd) II (oxyferryl heme intermediate) in the reaction of resting KatG[Y229F] with peroxyacetic acid or chloroperoxybenzoic acid, without detectable accumulation of Cmpd I (oxyferryl heme pi-cation radical intermediate), the latter being readily observed in the wild-type enzyme under similar conditions. Facile formation of Cmpd III (oxyferrous enzyme) also occurred in the mutant in the presence of micromolar hydrogen peroxide. Thus, the lost catalase function may be explained in part because of formation of intermediates that do not participate in catalatic turnover. The source of the reducing equivalent required for generation of Cmpd II from Cmpd I was shown by rapid freeze-quench electron paramagnetic resonance spectroscopy to be a tyrosine residue, just as in wild-type KatG. The kinetic coupling of radical generation and Cmpd II formation was shown in KatG[Y229F]. Residue Y229, which is a component of a newly defined three amino acid adduct in catalase-peroxidases, is critically important for protecting the catalase activity of KatG.
Highlights
Mycobacterium tuberculosis catalase-peroxidase (KatG)1 is a dimeric dual-function Class I heme peroxidase [1] of special interest in the field of tuberculosis research because of its role in the activation of the important antibiotic isoniazid and in the origins of resistance to this drug because of mutations in KatG
The formation of Cmpd III in the presence of H2O2 indicates that KatG enzymes must cycle through Cmpd II, because the expected pathway to oxyferrous enzyme is through an initial single-electron reaction of Cmpd II with hydrogen peroxide [15]
Characterization of Purified KatG[Y229F]—The WT KatG and KatG[Y229F] mutant enzymes were produced in E. coli using a previously described overexpression system carrying the wild-type M. tuberculosis katG gene or the mutated gene [9]
Summary
KatG, catalase-peroxidase; CcP, cytochrome c peroxidase; KatG[Y229F], Y229F mutant of KatG; Cmpd I, compound I (oxyferryl iron-protoporphyrin IX/-cation radical species); Cmpd II, compound II (oxyferryl heme intermediate); Cmpd III, compound III (oxyperoxidase); WT, wild-type; INH, isonicotinic acid hydrazide; PAA, peroxyacetic acid; CPBA, 3-chloroperoxybenzoic acid; EPR, electron paramagnetic resonance. KatGs have high homology to yeast cytochrome c peroxidase (CcP), including conserved distal arginine, tryptophan, and histidine residues and conserved proximal histidine and its hydrogen bonding partner, asparagine [2] Because these conserved residues are of key importance in governing catalytic mechanisms, strong functional homology would be expected among the Class I enzymes. With the first reports of the three-dimensional x-ray crystal structures of two bacterial catalase-peroxidases came the discovery of an unusual three-amino acid adduct involving covalently linked distal side tryptophan, tyrosine, and methionine residues [11, 12]. The presence of this adduct, may be the key feature that distinguishes KatGs from their homologues (Fig. 1). Rapid formation of Cmpd II and a tyrosyl radical, which disrupts catalase but not peroxidase function, is shown
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