title: Origin of Stereoselectivity and Substrate/Ligand Recognition in an FAD-Dependent R-Selective Amine Oxidase.
abstract: Elucidation of the molecular mechanism of amine oxidases (AOx) will help to extend their reactivity by rational design and their application to deracemization of various amine compounds. To date, several studies have been performed on S-selective AOx, but relatively few have focused on R-selective AOx. In this study, we sought to elucidate the mechanism of pkAOx, an R-selective AOx that we designed by introducing the Y228L and R283G mutations into d-amino acid oxidase from pig kidney. Four crystal structures of the substrate-bound protein and first-principles calculations based on the correlated fragment molecular orbital (FMO) indicated that two aromatic residues, Tyr224 and Phe242, form stable π-π stacking interaction with substrates. Enzyme kinetics also supported the importance of Tyr224 in catalysis: the kcat/Kmvalue of the Y224L mutant was reduced by 300-fold than that of wild-type (WT) when utilizing either (R)-methylbenzylamine [(R)-MBA] or (R)-1-(2-naphthyl)ethylamine [(R)-NEA] as the substrate. On the other hand, several Phe242 mutants exhibited higher reactivity toward (R)-NEA than the WT enzyme. In addition, FMO analysis indicated that pkAOx forms ∼13 kcal/mol more stable interaction with (R)-MBA than with (S)-MBA; this energy difference contributes to specific recognition of (R)-MBA in the racemate. Through the present study, we clarified three features of pkAOx: the roles of Tyr224 and Phe242 in catalysis, the origin of high stereoselectivity, and the potential to extend its reactivity toward amine compounds with bulky groups.
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.|School of Food and Nutritional Sciences, University of Shizuoka , 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.|Asano Active Enzyme Molecule Project, ERATO, JST , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Nakano
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.|Asano Active Enzyme Molecule Project, ERATO, JST , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Yasukawa
authors.affiliation: Department of Chemistry, Graduate School of Science, Tohoku University , Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
authors.forename: Takaki
authors.initials: T
authors.lastname: Tokiwa
authors.affiliation: Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University , 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ishikawa
authors.affiliation: Department of Chemistry, Rikkyo University , Nishi-ikebukuro, Toshimaku, Tokyo 171-8501, Japan.
authors.forename: Erika
authors.initials: E
authors.lastname: Ishitsubo
authors.affiliation: Department of Chemistry, Rikkyo University , Nishi-ikebukuro, Toshimaku, Tokyo 171-8501, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Matsuo
authors.affiliation: School of Food and Nutritional Sciences, University of Shizuoka , 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
authors.forename: Sohei
authors.initials: S
authors.lastname: Ito
authors.affiliation: Department of Chemistry, Rikkyo University , Nishi-ikebukuro, Toshimaku, Tokyo 171-8501, Japan.|Research Center of Smart Molecules, Rikkyo University , Nishi-ikebukuro, Toshimaku, Tokyo 171-8501, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tokiwa
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.|Asano Active Enzyme Molecule Project, ERATO, JST , 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano