Open AccessMutational and crystallographic analysis of l ‐amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities
Author(s) -
Matsui Daisuke,
Im Do-Hyun,
Sugawara Asami,
Fukuta Yasuhisa,
Fushinobu Shinya,
Isobe Kimiyasu,
Asano Yasuhisa
Publication year - 2014
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1016/j.fob.2014.02.002
Subject(s) - monooxygenase , chemistry , oxidase test , biochemistry , saturated mutagenesis , enzyme , amino acid , mutant , stereochemistry , mutagenesis , substrate (aquarium) , biology , cytochrome p450 , gene , ecology
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.