title: Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met-Tyr-Trp cross-linked adduct.
abstract: Mycobacterium tuberculosis catalase-peroxidase (Mtb KatG) is a bifunctional enzyme that possesses both catalase and peroxidase activities and is responsible for the activation of the antituberculosis drug isoniazid. Mtb KatG contains an unusual adduct in its distal heme-pocket that consists of the covalently linked Trp107, Tyr229, and Met255. The KatG(Y229F) mutant lacks this adduct and has decreased steady-state catalase activity and enhanced peroxidase activity. In order to test a potential structural role of the adduct that supports catalase activity, we have used resonance Raman spectroscopy to probe the local heme environment of KatG(Y229F). In comparison to wild-type KatG, resting KatG(Y229F) contains a significant amount of 6-coordinate, low-spin heme and a more planar heme. Resonance Raman spectroscopy of the ferrous-CO complex of KatG(Y229F) suggest a non-linear Fe-CO binding geometry that is less tilted than in wild-type KatG. These data provide evidence that the Met-Tyr-Trp adduct imparts structural stability to the active site of KatG that seems to be important for sustaining catalase activity.
authors.affiliation: Department of Chemistry, New York University, 100 Washington Square East, Room 1001, New York, NY 10003, USA.
authors.forename: Sofia M
authors.initials: SM
authors.lastname: Kapetanaki
authors.forename: Xiangbo
authors.initials: X
authors.lastname: Zhao
authors.forename: Shengwei
authors.initials: S
authors.lastname: Yu
authors.forename: Richard S
authors.initials: RS
authors.lastname: Magliozzo
authors.forename: Johannes P M
authors.initials: JP
authors.lastname: Schelvis
mesh_terms: D019943:Amino Acid Substitution; D000995:Antitubercular Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002374:Catalase / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D007538:Isoniazid / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D010544:Peroxidases / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D010544:Peroxidases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; D007538:Isoniazid