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

Ueda, Yuka; Yano, Yoshika; Nakayama, Natsume; Takekawa, Norihiro; Inagaki, Kenji; Imada, Katsumi

doi:https://doi.org/10.1002/pro.70432

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

アクセス数:15件（2026-02-15 20:41 集計）

固定URL: https://hdl.handle.net/11094/103713

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タイトル	Substrate recognition mechanisms of ʟ-glutamate oxidase from Streptomyces sp. and its conversion to ʟ-tyrosine oxidase
著者	著者名 Ueda, Yuka
	著者名 Yano, Yoshika
	著者名 Nakayama, Natsume
	NRID 1000050791810 1000050791810 ORCID 0000-0003-2802-4605 0000-0003-2802-4605 researchmap permalink takekawan takekawan Scopus Author ID 54416513800 54416513800 著者名 Takekawa, Norihiro
	NRID 1000080184711 1000080184711 著者名 Inagaki, Kenji
	NRID 1000040346143 1000040346143 ORCID 0000-0003-1342-8885 0000-0003-1342-8885 researchmap permalink read0096832 read0096832 Scopus Author ID 7006628770 7006628770 著者名 Imada, Katsumi
キーワード等	crystal structure
	l-glutamate oxidase
	modification of substrate specificity
	substrate recognition
内容	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.
抄録	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.
出版者	John Wiley and Sons Inc
収録物名	Protein Science
巻(号)	35(1)
ページ	e70432
刊行年月	2025-12-23
言語	英語
ハンドルURL	https://hdl.handle.net/11094/103713
PISSN	09618368
eISSN	1469896X
関連情報 (isIdenticalTo)	DOI (出版社版) https://doi.org/10.1002/pro.70432
アクセス権	open access
権利情報	This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
出版タイプ	Version of Record
カテゴリ	学術雑誌論文 Journal Article

資源タイプ	学術雑誌論文
ローカル資源タイプ	学術雑誌論文
DCMI資源タイプ	text
DCTERMS.bibliographicCitation	Protein Science. 2025, 35(1), p. e70432
DC.title	Substrate recognition mechanisms of ʟ-glutamate oxidase from Streptomyces sp. and its conversion to ʟ-tyrosine oxidase
DC.creator	Ueda, Yuka
	Yano, Yoshika
	Nakayama, Natsume
	Takekawa, Norihiro
	Inagaki, Kenji
	Imada, Katsumi
DC.publisher	John Wiley and Sons Inc
DC.language' scheme='DCTERMS.RFC1766	英語
DC.type' scheme='DCTERMS.DCMIType	text
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2025-12-23
DC.identifier	https://doi.org/10.1002/pro.70432
DC.subject	crystal structure
	l-glutamate oxidase
	modification of substrate specificity
	substrate recognition
DC.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.
DCTERMS.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.
DC.rights	This article is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
DC.format	103713
citation_title	Substrate recognition mechanisms of ʟ-glutamate oxidase from Streptomyces sp. and its conversion to ʟ-tyrosine oxidase
citation_author	Ueda, Yuka
	Yano, Yoshika
	Nakayama, Natsume
	Takekawa, Norihiro
	Inagaki, Kenji
	Imada, Katsumi
citation_publisher	John Wiley and Sons Inc
citation_language	英語
citation_date	2025-12-23
citation_journal_title	Protein Science
citation_volume	35
citation_issue	1
citation_firstpage	e70432
citation_doi	https://doi.org/10.1002/pro.70432
citation_public_url	https://hdl.handle.net/11094/103713

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

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