title: Crystal structure of aldoxime dehydratase and its catalytic mechanism involved in carbon-nitrogen triple-bond synthesis.
abstract: Aldoxime dehydratase (OxdA), which is a unique heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. Unlike the utilization of H(2)O(2) or O(2) as a mediator of catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron. Here, we determined the crystal structure of OxdA. We then constructed OxdA mutants in which each of the polar amino acids lying within ∼6 Å of the iron atom of the heme was converted to alanine. Among the purified mutant OxdAs, S219A had completely lost and R178A exhibited a reduction in the activity. Together with this finding, the crystal structural analysis of OxdA and spectroscopic and electrostatic potential analyses of the wild-type and mutant OxdAs suggest that S219 plays a key role in the catalysis, forming a hydrogen bond with the substrate. Based on the spatial arrangement of the OxdA active site and the results of a series of mutagenesis experiments, we propose the detailed catalytic mechanism of general aldoxime dehydratases: (i) S219 stabilizes the hydroxy group of the substrate to increase its basicity; (ii) H320 acts as an acid-base catalyst; and (iii) R178 stabilizes the heme, and would donate a proton to and accept one from H320.
authors: Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences,  University of Tsukuba, Tsukuba, Ibaraki 305-8572,  Japan.
authors: Junpei
authors: J
authors: Nomura
authors: Hiroshi
authors: H
authors: Hashimoto
authors: Takehiro
authors: T
authors: Ohta
authors: Yoshiteru
authors: Y
authors: Hashimoto
authors: Koichi
authors: K
authors: Wada
authors: Yoshinori
authors: Y
authors: Naruta
authors: Ken-Ichi
authors: K
authors: Oinuma
authors: Michihiko
authors: M
authors: Kobayashi
mesh_terms: D055162:Biocatalysis; D002244:Carbon / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D002244:Carbon; D006836:Hydro-Lyases; C412124:aldoxime dehydratase; D009584:Nitrogen
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