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| Main Authors: | , , , , , , , , |
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| Format: | Artículo científico |
| Language: | en |
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Biochemical and biophysical research communications
2026
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| Subjects: | |
| Online Access: | https://pubmed.ncbi.nlm.nih.gov/42229173/ |
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| _version_ | 1868266041737478145 |
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| author | Wu, Danni Zhang, Keke Luo, Quan Zhao, Kun Xu, Huifang Feng, Dandan Liang, Bo Ma, Honglei Lu, Xuefeng |
| author_facet | Wu, Danni Zhang, Keke Luo, Quan Zhao, Kun Xu, Huifang Feng, Dandan Liang, Bo Ma, Honglei Lu, Xuefeng Wu, Danni Zhang, Keke Luo, Quan Zhao, Kun Xu, Huifang Feng, Dandan Liang, Bo Ma, Honglei Lu, Xuefeng |
| collection | PubMed - marine biology |
| contents | Structural insights into flexible pyruvate binding in an (S)-selective ω-transaminase. Wu, Danni Zhang, Keke Luo, Quan Zhao, Kun Xu, Huifang Feng, Dandan Liang, Bo Ma, Honglei Lu, Xuefeng Pyruvic Acid Transaminases Crystallography, X-Ray Protein Binding Molecular Docking Simulation Substrate Specificity Protein Conformation Catalytic Domain Models, Molecular Binding Sites Pyridoxal Phosphate (S)-selective ω-transaminases (S-ωTAs) are PLP-dependent enzymes widely employed in biocatalysis for the stereoselective amination of prochiral ketones, yielding enantiopure (S)-amines. Although their stereochemical preference is well established, the structural basis of keto-acceptor recognition and active-site flexibility remains poorly understood. Here, we present a 1.96 Å crystal structure of a marine S-ωTA OM-S25 in complex with PLP and pyruvate (PYR). The enzyme exhibits four protomer in one asymmetric unit, yet electron density reveals pronounced conformational heterogeneity in PYR binding across protomers, channel-proximal transitional states (chain C), deeply buried productive poses (chains A, B, D), and a channel-entrance pose salt-bridged to Lys166. These conformations may represent a potential substrate binding route for acidic acceptors. Molecular docking corroborates the final PYR position, and a conserved flipping arginine, which usual refer to an arginine switch, stabilizes the carboxylate moiety of PYR-like substrates. We propose a stepwise entry pathway for the acceptor PYR in the second half-reaction of S-ωTAs. This pathway involves initial capture of PYR by Lys166 within the access channel, followed by an ∼180° rotation facilitated by Phe22, Tyr153, and Tyr168, progressive relocation through positions C→ D→A→B, and ultimate in the position of chain B for further reaction. Supporting evidence includes activity assays with PYR derivatives, thermal shift assays revealing modest stability perturbations, and gate-region mutagenesis experiments that confirm the proposed entry route. Although PMP-bound structures are essential to fully resolve the second half-reaction, this study provides the most comprehensive structural framework to date for acceptor recognition and the overall transamination mechanism in S-ωTAs. These findings lay a strong foundation for future mechanistic studies and rational enzyme engineering to advance biocatalytic applications. |
| format | Artículo científico |
| id | pubmed_42229173 |
| institution | PubMed |
| language | en |
| publishDate | 2026 |
| publisher | Biochemical and biophysical research communications |
| record_format | pubmed |
| spellingShingle | Structural insights into flexible pyruvate binding in an (S)-selective ω-transaminase. Wu, Danni Zhang, Keke Luo, Quan Zhao, Kun Xu, Huifang Feng, Dandan Liang, Bo Ma, Honglei Lu, Xuefeng Pyruvic Acid Transaminases Crystallography, X-Ray Protein Binding Molecular Docking Simulation Substrate Specificity Protein Conformation Catalytic Domain Models, Molecular Binding Sites Pyridoxal Phosphate Structural insights into flexible pyruvate binding in an (S)-selective ω-transaminase. Wu, Danni Zhang, Keke Luo, Quan Zhao, Kun Xu, Huifang Feng, Dandan Liang, Bo Ma, Honglei Lu, Xuefeng Pyruvic Acid Transaminases Crystallography, X-Ray Protein Binding Molecular Docking Simulation Substrate Specificity Protein Conformation Catalytic Domain Models, Molecular Binding Sites Pyridoxal Phosphate (S)-selective ω-transaminases (S-ωTAs) are PLP-dependent enzymes widely employed in biocatalysis for the stereoselective amination of prochiral ketones, yielding enantiopure (S)-amines. Although their stereochemical preference is well established, the structural basis of keto-acceptor recognition and active-site flexibility remains poorly understood. Here, we present a 1.96 Å crystal structure of a marine S-ωTA OM-S25 in complex with PLP and pyruvate (PYR). The enzyme exhibits four protomer in one asymmetric unit, yet electron density reveals pronounced conformational heterogeneity in PYR binding across protomers, channel-proximal transitional states (chain C), deeply buried productive poses (chains A, B, D), and a channel-entrance pose salt-bridged to Lys166. These conformations may represent a potential substrate binding route for acidic acceptors. Molecular docking corroborates the final PYR position, and a conserved flipping arginine, which usual refer to an arginine switch, stabilizes the carboxylate moiety of PYR-like substrates. We propose a stepwise entry pathway for the acceptor PYR in the second half-reaction of S-ωTAs. This pathway involves initial capture of PYR by Lys166 within the access channel, followed by an ∼180° rotation facilitated by Phe22, Tyr153, and Tyr168, progressive relocation through positions C→ D→A→B, and ultimate in the position of chain B for further reaction. Supporting evidence includes activity assays with PYR derivatives, thermal shift assays revealing modest stability perturbations, and gate-region mutagenesis experiments that confirm the proposed entry route. Although PMP-bound structures are essential to fully resolve the second half-reaction, this study provides the most comprehensive structural framework to date for acceptor recognition and the overall transamination mechanism in S-ωTAs. These findings lay a strong foundation for future mechanistic studies and rational enzyme engineering to advance biocatalytic applications. |
| title | Structural insights into flexible pyruvate binding in an (S)-selective ω-transaminase. |
| topic | Pyruvic Acid Transaminases Crystallography, X-Ray Protein Binding Molecular Docking Simulation Substrate Specificity Protein Conformation Catalytic Domain Models, Molecular Binding Sites Pyridoxal Phosphate |
| url | https://pubmed.ncbi.nlm.nih.gov/42229173/ |