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.

journal_name

FEBS Open Bio

journal_title

FEBS open bio

authors

Matsui D,Im DH,Sugawara A,Fukuta Y,Fushinobu S,Isobe K,Asano Y

doi

10.1016/j.fob.2014.02.002

subject

Has Abstract

pub_date

2014-02-07 00:00:00

pages

220-8

issn

2211-5463

pii

S2211-5463(14)00012-6

journal_volume

4

pub_type

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