{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T03:35:43Z","timestamp":1790307343471,"version":"4.1.0"},"reference-count":54,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T00:00:00Z","timestamp":1760054400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Jiangsu Ocean University for start-up funds","award":["KQ19026"],"award-info":[{"award-number":["KQ19026"]}]},{"name":"Natural Science Foundation of The Jiangsu Higher Education Institutions of China","award":["24KJB150007"],"award-info":[{"award-number":["24KJB150007"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Chiral amines are vital structural motifs in pharmaceuticals and agrochemicals, where enantiomeric purity governs bioactivity and environmental behavior. We identified a novel (R)-selective amine transaminase (MwoAT) from Mycobacterium sp. via genome mining, which exhibits activity toward the synthesis of the chiral amine (R)-1-methyl-3-phenylpropylamine. The enzyme displayed optimal activity at pH 7.0 and 40 \u00b0C, with high thermostability and solvent tolerance. Using an AlphaFold3-guided semi-rational engineering strategy integrating molecular docking, alanine scanning, and saturation mutagenesis, residue L175 was pinpointed as critical for substrate binding. The resulting L175G variant exhibited a 2.1-fold increase in catalytic efficiency (kcat\/Km) and improved thermal stability. Applied to the asymmetric synthesis of (R)-1-methyl-3-phenylpropylamine\u2014a precursor for the antihypertensive drug dilevalol and potential scaffold for crop protection agents\u2014the mutant achieved 26.4% conversion with \u226599.9% ee. The enzyme also accepted several ketones relevant to agrochemical synthesis, underscoring its versatility. This work delivers an engineered biocatalyst for sustainable chiral amine production and demonstrates an AI-assisted protein engineering framework applicable to both medicinal and agricultural chemistry.<\/jats:p>","DOI":"10.3390\/biom15101435","type":"journal-article","created":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T13:47:08Z","timestamp":1760104028000},"page":"1435","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["AlphaFold-Guided Semi-Rational Engineering of an (R)-Amine Transaminase for Green Synthesis of Chiral Amines"],"prefix":"10.3390","volume":"15","author":[{"given":"Xiaole","family":"Yang","sequence":"first","affiliation":[{"name":"Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xia","family":"Tian","sequence":"additional","affiliation":[{"name":"Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruizhou","family":"Tang","sequence":"additional","affiliation":[{"name":"Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiahuan","family":"Li","sequence":"additional","affiliation":[{"name":"Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuning","family":"Zhang","sequence":"additional","affiliation":[{"name":"Jiangsu BestEnzymes Biotech Co., Ltd., Lianyungang 222005, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tingting","family":"Li","sequence":"additional","affiliation":[{"name":"Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005, China"},{"name":"Jiangsu Institute of Marine Resources Development, Lianyungang 222005, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"16","DOI":"10.14227\/DT100303P16","article-title":"Overview on Chirality and Applications of Stereo-Selective Dissolution Testing in the Formulation and Development Work","volume":"10","author":"Gohel","year":"2003","journal-title":"Dissol. Technol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1002\/anie.196603851","article-title":"Specification of Molecular Chirality","volume":"5","author":"Cahn","year":"1996","journal-title":"Angew. Chem. Int. Ed. Eng."},{"key":"ref_3","first-page":"1","article-title":"Pharmacokinetics and pharmacodynamics of enantiomers of ibuprofen and flurbiprofen after oral administration","volume":"35","author":"Suri","year":"1997","journal-title":"Int. J. Clin. Pharmacol. Ther."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1274","DOI":"10.1016\/j.envpol.2018.09.095","article-title":"Enantioselectivity in biotransformation and bioaccumulation processes of typical chiral contaminants","volume":"243","author":"Xu","year":"2018","journal-title":"Environ. Pollut."},{"key":"ref_5","first-page":"1108","article-title":"Research progress on the stereoselective bioactivity and toxicity of chiral pesticides","volume":"24","author":"Haoming","year":"2022","journal-title":"Chin. J. Pestic. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1975","DOI":"10.1021\/acs.chemmater.3c02859","article-title":"Enantioselective Separation of Agricultural Fungicides Based on Chiral Hybrid Nanochannel Membranes","volume":"36","author":"Xu","year":"2024","journal-title":"Chem. Mater."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1039\/C6GC02328B","article-title":"Transaminase biocatalysis: Optimization and application","volume":"19","author":"Guo","year":"2017","journal-title":"Green Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"993","DOI":"10.1021\/cs300116n","article-title":"\u03c9-Transaminases for the Production of Optically Pure Amines and Unnatural Amino Acids","volume":"2","author":"Mathew","year":"2012","journal-title":"ACS Catal."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1007\/s11244-013-0184-1","article-title":"Biocatalytic Approaches to the Synthesis of Enantiomerically Pure Chiral Amines","volume":"57","author":"Ghislieri","year":"2013","journal-title":"Top. Catal."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"9760","DOI":"10.1039\/C5CC01594D","article-title":"Directed evolution 2.0: Improving and deciphering enzyme properties","volume":"51","author":"Cheng","year":"2015","journal-title":"Chem. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2999","DOI":"10.1007\/s00253-020-10444-y","article-title":"Fluorescence-based high-throughput screening system for R-\u03c9-transaminase engineering and its substrate scope extension","volume":"104","author":"Cheng","year":"2020","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1201","DOI":"10.1021\/jacs.8b11561","article-title":"Biocatalytic N-Alkylation of Amines Using Either Primary Alcohols or Carboxylic Acids via Reductive Aminase Cascades","volume":"141","author":"Ramsden","year":"2019","journal-title":"J. Am. Chem. Soc."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"324","DOI":"10.1016\/j.tibtech.2010.03.003","article-title":"\u03c9-Transaminases for the synthesis of non-racemic \u03b1-chiral primary amines","volume":"28","author":"Koszelewski","year":"2010","journal-title":"Trends Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1016\/j.cbpa.2017.12.007","article-title":"Transaminases for chiral amine synthesis","volume":"43","author":"Gomm","year":"2018","journal-title":"Curr. Opin. Chem. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1021\/acs.chemrev.7b00437","article-title":"Application of \u03c9-Transaminases in the Pharmaceutical Industry","volume":"118","author":"Kelly","year":"2017","journal-title":"Chem. Rev."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"R1","DOI":"10.1016\/S0969-2126(00)00085-X","article-title":"The manifold of vitamin B6 dependent enzymes","volume":"8","author":"Schneider","year":"2000","journal-title":"Structure"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"8453","DOI":"10.1039\/C5OB00690B","article-title":"A quantum chemical study of the \u03c9-transaminase reaction mechanism","volume":"13","author":"Cassimjee","year":"2015","journal-title":"Org. Biomol. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"807","DOI":"10.1002\/adsc.200800030","article-title":"A Protection Strategy Substantially Enhances Rate and Enantioselectivity in \u03c9-Transaminase-Catalyzed Kinetic Resolutions","volume":"350","author":"Robins","year":"2008","journal-title":"Adv. Synth. Catal."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"246","DOI":"10.1016\/j.bej.2007.05.001","article-title":"Improved activity and thermostability of (S)-aminotransferase by error-prone polymerase chain reaction for the production of a chiral amine","volume":"37","author":"Martin","year":"2007","journal-title":"Biochem. Eng. J."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1016\/j.enzmictec.2007.05.011","article-title":"Substrate spectrum of \u03c9-transaminase from Chromobacterium violaceum DSM30191 and its potential for biocatalysis","volume":"41","author":"Kaulmann","year":"2007","journal-title":"Enzyme Microb. Technol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Xie, Z., Zhai, L., Meng, D., Tian, Q., Guan, Z., Cai, Y., and Liao, X. (2020). Improving the catalytic thermostability of Bacillus altitudinis W3 \u03c9-transaminase by proline substitutions. 3 Biotech, 10.","DOI":"10.1007\/s13205-020-02321-2"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1126\/science.1188934","article-title":"Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture","volume":"329","author":"Savile","year":"2010","journal-title":"Science"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1288","DOI":"10.1002\/cctc.201902056","article-title":"Three-liquid-phase Spinning Reactor for the Transaminase-catalyzed Synthesis and Recovery of a Chiral Amine","volume":"12","author":"Matassa","year":"2020","journal-title":"ChemCatChem"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1146\/annurev-biochem-060713-035600","article-title":"Biosynthesis and Export of Bacterial Lipopolysaccharides","volume":"83","author":"Whitfield","year":"2014","journal-title":"Annu. Rev. Biochem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1016\/S0959-440X(00)00106-8","article-title":"Tailoring new enzyme functions by rational redesign","volume":"10","author":"Cedrone","year":"2000","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"134429","DOI":"10.1016\/j.fuel.2025.134429","article-title":"Engineering an \u03c9-transaminase for chemoenzymatically synthesizing key intermediate 5-hydroxymethyl-2-furfurylamine","volume":"388","author":"Yang","year":"2025","journal-title":"Fuel"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1007\/s43393-020-00005-9","article-title":"Current advances in design and engineering strategies of industrial enzymes","volume":"1","author":"Dinmukhamed","year":"2020","journal-title":"Syst. Microbiol. Biomanuf."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"110505","DOI":"10.1016\/j.enzmictec.2024.110505","article-title":"Semi-rational engineering of \u03c9-transaminase for enhanced enzymatic activity to 2-ketobutyrate","volume":"180","author":"Zhang","year":"2024","journal-title":"Enzyme Microb. Technol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4141","DOI":"10.1021\/cr100386u","article-title":"Application of Designed Enzymes in Organic Synthesis","volume":"111","author":"Strohmeier","year":"2011","journal-title":"Chem. Rev."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1080\/17460441.2018.1403419","article-title":"Molecular dynamics simulations and novel drug discovery","volume":"13","author":"Liu","year":"2017","journal-title":"Expert. Opin. Drug Discov."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Zhu, W.-L., Hu, S., Lv, C.-J., Zhao, W.-R., Wang, H.-P., Mei, J.-Q., Mei, L.-H., and Huang, J. (2019). A Single Mutation Increases the Thermostability and Activity of Aspergillus terreus Amine Transaminase. Molecules, 24.","DOI":"10.3390\/molecules24071194"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2613","DOI":"10.1007\/s00253-014-6056-1","article-title":"Characterization of (R)-selective amine transaminases identified by in silico motif sequence blast","volume":"99","author":"Jiang","year":"2014","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e02338","DOI":"10.1016\/j.heliyon.2019.e02338","article-title":"Whole cell biosynthesis of 1-methyl-3-phenylpropylamine and 2-amino-1,3,4-butanetriol using Komagataella phaffii (Pichia pastoris) strain BG-10 engineered with a transgene encoding Chromobacterium violaceum \u03c9-transaminase","volume":"5","author":"Henriquez","year":"2019","journal-title":"Heliyon"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"499","DOI":"10.1007\/s00253-005-0002-1","article-title":"Microbial synthesis of chiral amines by (R)-specific transamination with Arthrobacter sp. KNK168","volume":"69","author":"Iwasaki","year":"2006","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"6614","DOI":"10.1093\/nar\/gkw569","article-title":"NCBI prokaryotic genome annotation pipeline","volume":"44","author":"Tatusova","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_37","first-page":"15","article-title":"Molecular Dynamics Simulations Studies Based GROMACS","volume":"29","author":"Jiang","year":"2016","journal-title":"Med. Inf."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1950","DOI":"10.1002\/prot.22711","article-title":"Improved side-chain torsion potentials for the Amber ff99SB protein force field","volume":"78","author":"Piana","year":"2010","journal-title":"Proteins Struct. Funct. Bioinform."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2587","DOI":"10.1021\/acs.jcim.5b00308","article-title":"Force-Field Induced Bias in the Structure of A\u03b221\u201330: A Comparison of OPLS, AMBER, CHARMM, and GROMOS Force Fields","volume":"55","author":"Smith","year":"2015","journal-title":"J. Chem. Inf. Model."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"100329","DOI":"10.1016\/j.chphi.2023.100329","article-title":"Dynamics of an inhibitor at and away from an out-of-active site binding location in human carbonic anhydrase II","volume":"7","author":"Khatua","year":"2023","journal-title":"Chem. Phys. Impact"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.biochi.2018.12.017","article-title":"Biochemical and structural insights into PLP fold type IV transaminase from Thermobaculum terrenum","volume":"158","author":"Bezsudnova","year":"2018","journal-title":"Biochimie"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Li, T., Zhu, H., Jia, W., Tian, X., Xu, Z., Zhu, J., Liu, W., and Cao, Y. (2024). Identification, characterization and application of M16AT, a new organic solvent-tolerant (R)-enantio-selective type IV amine transaminase from Mycobacterium sp. ACS1612. Chembiochem, 25.","DOI":"10.1002\/cbic.202300812"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1563","DOI":"10.1007\/s00253-011-3580-0","article-title":"A novel transaminase, (R)-amine:pyruvate aminotransferase, from Arthrobacter sp. KNK168 (FERM BP-5228): Purification, characterization, and gene cloning","volume":"93","author":"Iwasaki","year":"2011","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"809","DOI":"10.1021\/acs.accounts.5b00516","article-title":"Protein Binding Pocket Dynamics","volume":"49","author":"Stank","year":"2016","journal-title":"Acc. Chem. Res."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"W48","DOI":"10.1093\/nar\/gkaa235","article-title":"ProteinsPlus: Interactive analysis of protein\u2013ligand binding interfaces","volume":"48","author":"Diedrich","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1021\/acscatal.2c04735","article-title":"Reshaping Substrate-Binding Pocket of Leucine Dehydrogenase for Bidirectionally Accessing Structurally Diverse Substrates","volume":"13","author":"Wu","year":"2022","journal-title":"ACS Catal."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"9564","DOI":"10.1002\/anie.201100208","article-title":"Putting Anion\u2013\u03c0 Interactions Into Perspective","volume":"50","author":"Frontera","year":"2011","journal-title":"Angew. Chem. Int. Ed. Engl."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Hou, Y., Zou, L., Li, Q., Chen, M., Ruan, H., Sun, Z., Xu, X., Yang, J., and Ma, G. (2022). Supramolecular assemblies based on natural small molecules: Union would be effective. Mater. Today Bio, 15.","DOI":"10.1016\/j.mtbio.2022.100327"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Zhu, R.-G., Xiao, H.-Y., Zhou, Z., Yin, M., Xiao, H., Hu, C., Wei, G., and Liu, C. (2024). Thermal degradation of 18 amino acids during pyrolytic processes. Sci. Rep., 14.","DOI":"10.1038\/s41598-024-79032-8"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"3310","DOI":"10.1021\/jacsau.4c00649","article-title":"Connecting Protein Millisecond Conformational Dynamics to Protein Thermal Stability","volume":"4","author":"Hou","year":"2024","journal-title":"JACS Au"},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Meng, Q., Ram\u00edrez-Palacios, C., Wijma, H.J., and Janssen, D.B. (2022). Protein engineering of amine transaminases. Front. Catal., 2.","DOI":"10.3389\/fctls.2022.1049179"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"e00062-22","DOI":"10.1128\/aem.00062-22","article-title":"Engineering Novel (R)-Selective Transaminase for Efficient Symmetric Synthesis of d-Alanine","volume":"88","author":"Jia","year":"2022","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1002\/bit.25017","article-title":"A model to assess the feasibility of shifting reaction equilibrium by acetone removal in the transamination of ketones using 2-propylamine","volume":"111","author":"Tufvesson","year":"2013","journal-title":"Biotechnol. Bioeng."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"5798","DOI":"10.1039\/C4CC08752F","article-title":"Cascade catalysis\u2014Strategies and challenges en route to preparative synthetic biology","volume":"51","author":"Muschiol","year":"2015","journal-title":"Chem. Commun."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/10\/1435\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T04:12:05Z","timestamp":1760328725000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/15\/10\/1435"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,10]]},"references-count":54,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["biom15101435"],"URL":"https:\/\/doi.org\/10.3390\/biom15101435","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,10]]}}}