Evidence for radical formation at Tyr-353 in Mycobacterium tuberculosis catalase-peroxidase (KatG).
Mycobacterium tuberculosis KatG is a heme-containing catalase-peroxidase responsible for activation, through its peroxidase cycle, of the front line antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide). Formation of Compound I (oxyferryl heme-porphyrin pi-cation radical), the classical peroxidase intermediate generated when the resting enzyme turns over with alkyl peroxides, is rapidly followed by production of a protein-centered tyrosyl radical in this enzyme. In our efforts to identify the residue at which this radical is formed, nitric oxide was used as a radical scavenging reagent. Quenching of the tyrosyl radical generated in the presence of NO was shown using electron paramagnetic resonance spectroscopy, and formation of nitrotyrosine was confirmed by proteolytic digestion followed by high performance liquid chromatography analysis of the NO-treated enzyme. These results are consistent with formation of nitrosyltyrosine by addition of NO to tyrosyl radical and oxidation of this intermediate to nitrotyrosine. Two predominant nitrotyrosine-containing peptides were identified that were purified and sequenced by Edman degradation. Both peptides were derived from the same M. tuberculosis KatG sequence spanning residues 346-356 with the amino acid sequence SPAGAWQYTAK, and both peptides contained nitrotyrosine at residue 353. Some modification of Trp-351 most probably into nitrosotryptophan was also found in one of the two peptides. Control experiments using denatured KatG or carried out in the absence of peroxide did not produce nitrotyrosine. In the mutant enzyme KatG(Y353F), which was constructed using site-directed mutagenesis, a tyrosyl radical was also formed upon turnover with peroxide but in poor yield compared with wild-type KatG. Residue Tyr-353 is unique to M. tuberculosis KatG and may play a special role in the function of this enzyme.
Zhao
Xiangbo
X
Department of Chemistry, Brooklyn College of the City University of New York, Brooklyn, New York 11210, USA.
Girotto
Stefania
S
Yu
Shengwei
S
Magliozzo
Richard S
RS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002374:Catalase; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009569:Nitric Oxide / Q000737:chemistry; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry*
D001426:Bacterial Proteins; D005609:Free Radicals; D010545:Peroxides; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D014364:Tryptophan; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis