Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met-Tyr-Trp cross-linked adduct |
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Authors: | Kapetanaki Sofia M Zhao Xiangbo Yu Shengwei Magliozzo Richard S Schelvis Johannes P M |
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Institution: | Department of Chemistry, New York University, 100 Washington Square East, Room 1001, New York, NY 10003, USA. |
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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. |
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Keywords: | Mycobacterium tuberculosis Catalase-peroxidase Resonance Raman Site-directed mutagenesis |
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