title: Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities.
abstract: In this study, it was shown for the first time that l-amino acid oxidase of Pseudomonas sp. AIU813, renamed as l-amino acid oxidase/monooxygenase (l-AAO/MOG), exhibits l-lysine 2-monooxygenase as well as oxidase activity. l-Lysine oxidase activity of l-AAO/MOG was increased in a p-chloromercuribenzoate (p-CMB) concentration-dependent manner to a final level that was five fold higher than that of the non-treated enzyme. In order to explain the effects of modification by the sulfhydryl reagent, saturation mutagenesis studies were carried out on five cysteine residues, and we succeeded in identifying l-AAO/MOG C254I mutant enzyme, which showed five-times higher specific activity of oxidase activity than that of wild type. The monooxygenase activity shown by the C254I variant was decreased significantly. Moreover, we also determined a high-resolution three-dimensional structure of l-AAO/MOG to provide a structural basis for its biochemical characteristics. The key residue for the activity conversion of l-AAO/MOG, Cys-254, is located near the aromatic cage (Trp-418, Phe-473, and Trp-516). Although the location of Cys-254 indicates that it is not directly involved in the substrate binding, the chemical modification by p-CMB or C254I mutation would have a significant impact on the substrate binding via the side chain of Trp-516. It is suggested that a slight difference of the binding position of a substrate can dictate the activity of this type of enzyme as oxidase or monooxygenase.
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: Daisuke
authors.initials: D
authors.lastname: Matsui
authors.affiliation: Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Do-Hyun
authors.initials: DH
authors.lastname: Im
authors.affiliation: Department of Biological Chemistry and Food Science, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan.
authors.forename: Asami
authors.initials: A
authors.lastname: Sugawara
authors.affiliation: Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Fukuta
authors.affiliation: Asano Active Enzyme Molecule Project, ERATO, JST, 5180 Kurokawa, Imizu,Toyama 939-0398, Japan ; Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Fushinobu
authors.affiliation: Department of Biological Chemistry and Food Science, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan.
authors.forename: Kimiyasu
authors.initials: K
authors.lastname: Isobe
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
keywords: 4-AA, 4-aminoantipyrine; CHCA, α-Cyano-4-hydroxycinnamic acid; Crystallography; FMOs, flavin monooxygenases; Flavin monooxygenases; Flavin-containing monoamine oxidase family; LB, Luria–Bertani; LGOX, l-glutamate oxidase; MAO, flavin-containing monoamine oxidase; PAO, l-phenylalanine oxidase; Saturation mutagenesis; TFA, trifluoroacetic acid; TMO, l-tryptophan 2-monooxygenase; TOOS, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline; amid, amide hydrolase gene; l-AAO, l-amino acid oxidase; l-AAO/MOG, l-amino acid oxidase/monooxygenase; l-Amino acid oxidase/monooxygenase; laao/mog, l-amino acid oxidase/monooxygenase gene; p-CMB, p-chloromercuribenzoate