Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction
Abstract Production of D-amino acids (D-AAs) on a large-scale enables to provide precursors of peptide therapeutics. In this study, we designed a novel L-amino acid oxidase, HTAncLAAO2, by ancestral sequence reconstruction, exhibiting high thermostability and long-term stability. The crystal structu...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-09-01
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| Series: | Communications Chemistry |
| Online Access: | https://doi.org/10.1038/s42004-023-01005-1 |
| _version_ | 1797577628762243072 |
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| author | Yui Kawamura Chiharu Ishida Ryo Miyata Azusa Miyata Seiichiro Hayashi Daisuke Fujinami Sohei Ito Shogo Nakano |
| author_facet | Yui Kawamura Chiharu Ishida Ryo Miyata Azusa Miyata Seiichiro Hayashi Daisuke Fujinami Sohei Ito Shogo Nakano |
| author_sort | Yui Kawamura |
| collection | DOAJ |
| description | Abstract Production of D-amino acids (D-AAs) on a large-scale enables to provide precursors of peptide therapeutics. In this study, we designed a novel L-amino acid oxidase, HTAncLAAO2, by ancestral sequence reconstruction, exhibiting high thermostability and long-term stability. The crystal structure of HTAncLAAO2 was determined at 2.2 Å by X-ray crystallography, revealing that the enzyme has an octameric form like a “ninja-star” feature. Enzymatic property analysis demonstrated that HTAncLAAO2 exhibits three-order larger k cat/K m values towards four L-AAs (L-Phe, L-Leu, L-Met, and L-Ile) than that of L-Trp. Through screening the variants, we obtained the HTAncLAAO2(W220A) variant, which shows a > 6-fold increase in k cat value toward L-Trp compared to the original enzyme. This variant applies to synthesizing enantio-pure D-Trp derivatives from L- or rac-forms at a preparative scale. Given its excellent properties, HTAncLAAO2 would be a starting point for designing novel oxidases with high activity toward various amines and AAs. |
| first_indexed | 2024-03-10T22:10:46Z |
| format | Article |
| id | doaj.art-df1371eaef6541c18fdebc8e1b4aa00f |
| institution | Directory Open Access Journal |
| issn | 2399-3669 |
| language | English |
| last_indexed | 2024-03-10T22:10:46Z |
| publishDate | 2023-09-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Chemistry |
| spelling | doaj.art-df1371eaef6541c18fdebc8e1b4aa00f2023-11-19T12:37:07ZengNature PortfolioCommunications Chemistry2399-36692023-09-016111010.1038/s42004-023-01005-1Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reactionYui Kawamura0Chiharu Ishida1Ryo Miyata2Azusa Miyata3Seiichiro Hayashi4Daisuke Fujinami5Sohei Ito6Shogo Nakano7Graduate Division of Nutritional and Environmental Sciences, University of ShizuokaGraduate Division of Nutritional and Environmental Sciences, University of ShizuokaGraduate Division of Nutritional and Environmental Sciences, University of ShizuokaGraduate Division of Nutritional and Environmental Sciences, University of ShizuokaDivision of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Higashi-kuGraduate Division of Nutritional and Environmental Sciences, University of ShizuokaGraduate Division of Nutritional and Environmental Sciences, University of ShizuokaGraduate Division of Nutritional and Environmental Sciences, University of ShizuokaAbstract Production of D-amino acids (D-AAs) on a large-scale enables to provide precursors of peptide therapeutics. In this study, we designed a novel L-amino acid oxidase, HTAncLAAO2, by ancestral sequence reconstruction, exhibiting high thermostability and long-term stability. The crystal structure of HTAncLAAO2 was determined at 2.2 Å by X-ray crystallography, revealing that the enzyme has an octameric form like a “ninja-star” feature. Enzymatic property analysis demonstrated that HTAncLAAO2 exhibits three-order larger k cat/K m values towards four L-AAs (L-Phe, L-Leu, L-Met, and L-Ile) than that of L-Trp. Through screening the variants, we obtained the HTAncLAAO2(W220A) variant, which shows a > 6-fold increase in k cat value toward L-Trp compared to the original enzyme. This variant applies to synthesizing enantio-pure D-Trp derivatives from L- or rac-forms at a preparative scale. Given its excellent properties, HTAncLAAO2 would be a starting point for designing novel oxidases with high activity toward various amines and AAs.https://doi.org/10.1038/s42004-023-01005-1 |
| spellingShingle | Yui Kawamura Chiharu Ishida Ryo Miyata Azusa Miyata Seiichiro Hayashi Daisuke Fujinami Sohei Ito Shogo Nakano Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction Communications Chemistry |
| title | Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction |
| title_full | Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction |
| title_fullStr | Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction |
| title_full_unstemmed | Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction |
| title_short | Structural and functional analysis of hyper-thermostable ancestral L-amino acid oxidase that can convert Trp derivatives to D-forms by chemoenzymatic reaction |
| title_sort | structural and functional analysis of hyper thermostable ancestral l amino acid oxidase that can convert trp derivatives to d forms by chemoenzymatic reaction |
| url | https://doi.org/10.1038/s42004-023-01005-1 |
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