title: Role of the Met-Tyr-Trp cross-link in Mycobacterium tuberculosis catalase-peroxidase (KatG) as revealed by KatG(M255I).
abstract: Catalase-peroxidases (KatGs) are bifunctional enzymes possessing both catalase and peroxidase activities. Four crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link which has been suggested to impart catalatic activity to the KatGs. To decipher the individual roles of the two cross-links in the Met-Tyr-Trp adduct, we have focused on recombinant Mycobacterium tuberculosis KatG(M255I). UV-visible spectroscopic and mass spectrometric studies of the peptide fragments resulting from tryptic digestion of KatG(M255I) confirmed the presence of the single Tyr-Trp cross-link, as well as a 2e- oxidized form which is postulated to be an intermediate generated during Met-Tyr-Trp cross-link formation. KatG(M255I) lacking the Tyr-Trp cross-link was also prepared, and incubation with peroxyacetic acid, but not 2-methyl-1-phenyl-2-propyl hydroperoxide, resulted in complete formation of the Tyr-Trp cross-link. A mechanism for Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies with KatG(M255I) were performed, allowing characterization of compounds I, II, and III. Interestingly, two compound II intermediates were identified: (KatG*)(Por)Fe(III)-OH, where KatG* represents a protein-based radical, and oxoferryl (KatG)(Por)Fe(IV)=O. Insight into the contributions of the individual Tyr-Trp and Met-Tyr cross-links to catalase activity is presented, as is the overall contribution of the Met-Tyr-Trp cross-link to the structure-function-spectroscopy relationship and catalase-peroxidase mechanism in KatG.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-2280, USA.
authors.forename: Reza A
authors.initials: RA
authors.lastname: Ghiladi
authors.forename: Katalin F
authors.initials: KF
authors.lastname: Medzihradszky
authors.forename: Paul R
authors.initials: PR
authors.lastname: Ortiz de Montellano
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002374:Catalase / Q000737:chemistry* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010463:Peracetic Acid / Q000737:chemistry; D010544:Peroxidases / Q000032:analysis; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D014443:Tyrosine; D014364:Tryptophan; D008715:Methionine; D010544:Peroxidases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D010463:Peracetic Acid