title: Substrate-assisted O2 activation in a cofactor-independent dioxygenase.
abstract: In contrast to the majority of O2-activating enzymes, which depend on an organic cofactor or a metal ion for catalysis, a particular group of structurally unrelated oxygenases is functional without any cofactor. In this study, we characterized the mechanism of O2 activation in the reaction pathway of a cofactor-independent dioxygenase with an α/β-hydrolase fold, which catalyzes the oxygenolytic cleavage of 2-alkyl-3-hydroxy-4(1H)-quinolones. Chemical analysis and electron paramagnetic resonance spectroscopic data revealed that O2 activation in the enzyme's active site is substrate-assisted, relying on single electron transfer from the bound substrate anion to O2 to form a radical pair, which recombines to a C2-peroxide intermediate. Thus, an oxygenase can function without a cofactor, if the organic substrate itself, after activation to a (carb)anion by an active-site base, is intrinsically reactive toward molecular oxygen.
authors.affiliation: Institute of Molecular Microbiology and Biotechnology, University of Münster, Corrensstrasse 3, 48149 Münster, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Thierbach
authors.affiliation: Department of Biophysics, School of Medicine, Saarland University, Clinical Center, Building 76, 66421 Homburg, Germany.
authors.forename: Nguyen
authors.initials: N
authors.lastname: Bui
authors.affiliation: Pharmaceutical Biology, Saarland University, Campus, Building C2, 66123 Saarbrücken, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Zapp
authors.affiliation: School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
authors.forename: Siri Ram
authors.initials: SR
authors.lastname: Chhabra
authors.affiliation: Department of Biophysics, School of Medicine, Saarland University, Clinical Center, Building 76, 66421 Homburg, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Kappl
authors.affiliation: Institute of Molecular Microbiology and Biotechnology, University of Münster, Corrensstrasse 3, 48149 Münster, Germany. Electronic address: fetzner@uni-muenster.de.
authors.forename: Susanne
authors.initials: S
authors.lastname: Fetzner
mesh_terms: D019943:Amino Acid Substitution; D055162:Biocatalysis; D020134:Catalytic Domain; D049308:Dioxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology; D015363:Quinolones / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D015363:Quinolones; D006861:Hydrogen Peroxide; C101932:1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (decyclizing, CO-forming); D049308:Dioxygenases; D010100:Oxygen