Dioxygenases Without Requirement for Cofactors: Identification of Amino Acid Residues Involved in Substrate Binding and Catalysis, and Testing for Rate-Limiting Steps in the Reaction of 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase |
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Authors: | Ursula Frerichs-Deeken Susanne Fetzner |
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Affiliation: | 1. Institut für Molekulare Mikrobiologie und Biotechnologie, Westf?lische Wilhelms-Universit?t Münster, Münster, D-48149, Germany 2. AG Mikrobiologie, Institut für Chemie und Biologie des Meeres, Carl von Ossietzky Universit?t Oldenburg, Oldenburg, D-26111, Germany
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Abstract: | 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod), catalyzing cleavage of its heteroaromatic substrate to form carbon monoxide and N-acetylanthranilate, belongs to the α/β hydrolase fold family of enzymes. Analysis of protein variants suggested that Hod has adapted active-site residues of the α/β hydrolase fold for the dioxygenolytic reaction. H251 was recently shown to act as a general base to abstract a proton from the organic substrate. Residue S101, which corresponds to the nucleophile of the catalytic triad of α/β-hydrolases, presumably participates in binding the heteroaromatic substrate. H102 and residues located in the topological region of the triad’s acidic residue appear to influence O2 binding and reactivity. A tyrosine residue might be involved in the turnover of the ternary complex [HodH+–3,4-dioxyquinaldine dianion–O2]. Absence of viscosity effects and kinetic solvent isotope effects suggests that turnover of the ternary complex, rather than substrate binding, product release, or proton movements, involves the rate-determining step in the reaction catalyzed by Hod. |
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