Substrate recognition mechanisms of ʟ-glutamate oxidase from Streptomyces sp. and its conversion to ʟ-tyrosine oxidase

Ueda, Yuka; Yano, Yoshika; Nakayama, Natsume et al.

Protein Science, 2025, 35(1), e70432

Number of Access:17（2026-02-18 23:53 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/103713

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Title
Substrate recognition mechanisms of ʟ-glutamate oxidase from Streptomyces sp. and its conversion to ʟ-tyrosine oxidase
Creator
CreatorName
Ueda, Yuka
CreatorName
Yano, Yoshika
CreatorName
Nakayama, Natsume
NRID
1000050791810 1000050791810
ORCID
0000-0003-2802-4605 0000-0003-2802-4605
researchmap permalink
takekawan takekawan
Scopus Author ID
54416513800 54416513800
CreatorName
Takekawa, Norihiro
NRID
1000080184711 1000080184711
CreatorName
Inagaki, Kenji
NRID
1000040346143 1000040346143
ORCID
0000-0003-1342-8885 0000-0003-1342-8885
researchmap permalink
read0096832 read0096832
Scopus Author ID
7006628770 7006628770
CreatorName
Imada, Katsumi
Subject
crystal structure
l-glutamate oxidase
modification of substrate specificity
substrate recognition
Description
Ueda Y., Yano Y., Nakayama N., et al. Substrate recognition mechanisms of ʟ-glutamate oxidase from Streptomyces sp. and its conversion to ʟ-tyrosine oxidase. Protein Science 35, e70432 (2026); https://doi.org/10.1002/pro.70432.
Abstract
ʟ-Amino acid oxidase (LAAO) is a flavoenzyme that catalyzes the oxidative deamination of ʟ-amino acids, producing α-keto acids, ammonia, and hydrogen peroxide. Among LAAOs, ʟ-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 exhibits exceptionally high substrate specificity for ʟ-glutamate. LGOX is expressed as a homodimeric precursor and undergoes proteolytic processing for maturation. Structural studies revealed that LGOX comprises an FAD-binding domain, a substrate-binding domain, and a helical domain. Conserved residues W653, R124, and Y562 that recognize the α-amino and α-carboxyl groups of the substrate exist in the putative active site. R305 was identified as a key determinant for side-chain recognition; its substitution with Glu conferred specific activity toward ʟ-arginine, effectively converting LGOX into an ʟ-arginine oxidase. However, the putative substrate binding pocket includes an acidic residue, E617, undesirable for acidic substrates. Therefore, the mechanism of high specificity for ʟ-glutamate remains unclear. To elucidate the molecular basis for the high substrate specificity of LGOX, we determined the structure of LGOX in complex with ʟ-glutamate. Structural and mutational analyses revealed that E617 plays a critical role in substrate binding by aligning the side chain of R305. The loop at the entrance of the tunnel to the substrate-binding site regulates the access of the substrate to the site. Furthermore, E617F and E617K variants acquired ʟ-tyrosine oxidase activity, providing insight into how specificity can be redirected. These findings clarify the substrate recognition mechanism of LGOX and underscore its potential as a robust scaffold for engineering novel amino acid oxidases with tailored specificities.
Publisher
John Wiley and Sons Inc
Source Title
Protein Science
Volume (Issue)
35(1)
Page
e70432
Date of Issued
2025-12-23
Language
English
Handle URL
https://hdl.handle.net/11094/103713
PISSN
09618368
eISSN
1469896X
Relation.isIdenticalTo
DOI (Publisher Version)
https://doi.org/10.1002/pro.70432
Access Rights
open access
CopyRight
This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
oaire:version
Version of Record
Category
学術雑誌論文 Journal Article