Evolving ω-amine transaminase At ATA guided by substrate-enzyme binding free energy for enhancing activity and stability against non-natural substrates

• Published in: Applied and environmental microbiology 2024-07, Vol.90 (7), p.e0054324
• Main Authors: Qiu, Shuai, Ju, Cong-Lin, Wang, Tong, Chen, Jie, Cui, Yu-Tong, Wang, Lin-Quan, Fan, Fang-Fang, Huang, Jun
• Format: Article
• Language: English
• Subjects: Amines - chemistry; Amines - metabolism; Aspergillus - enzymology; Aspergillus - genetics; Catalytic Domain; Enzyme Stability; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Substrate Specificity; Transaminases - chemistry; Transaminases - genetics; Transaminases - metabolism
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.00543-24

In the field of chiral amine synthesis, ω-amine transaminase (ω-ATA) is one of the most established enzymes capable of asymmetric amination under optimal conditions. However, the applicability of ω-ATA toward more non-natural complex molecules remains limited due to its low transamination activity, thermostability, and narrow substrate scope. Here, by employing a combined approach of computational virtual screening strategy and combinatorial active-site saturation test/iterative saturation mutagenesis strategy, we have constructed the best variant M14C3-V5 (M14C3-V62A-V116S-E117I-L118I-V147F) with improved ω-ATA from ( ATA) activity and thermostability toward non-natural substrate 1-acetylnaphthalene, which is the ketone precursor for producing the intermediate ( )-(+)-1-(1-naphthyl)ethylamine [( )-NEA] of cinacalcet hydrochloride, showing activity enhancement of up to 3.4-fold compared to parent enzyme M14C3 ( ATA-F115L-M150C-H210N-M280C-V149A-L182F-L187F). The computational tools YASARA, Discovery Studio, Amber, and FoldX were applied for predicting mutation hotspots based on substrate-enzyme binding free energies and to show the possible mechanism with features related to ATA structure, catalytic activity, and stability analyses. M14C3-V5 achieved 71.8% conversion toward 50 mM 1-acetylnaphthalene in a 50 mL preparative-scale reaction for preparing ( )-NEA. Moreover, M14C3-V5 expanded the substrate scope toward aromatic ketone compounds. The generated virtual screening strategy based on the changes in binding free energies has successfully predicted the ATA activity toward 1-acetylnaphthalene and related substrates. Together with experimental data, these approaches can serve as a gateway to explore desirable performances, expand enzyme-substrate scope, and accelerate biocatalysis.IMPORTANCEChiral amine is a crucial compound with many valuable applications. Their asymmetric synthesis employing ω-amine transaminases (ω-ATAs) is considered an attractive method. However, most ω-ATAs exhibit low activity and stability toward various non-natural substrates, which limits their industrial application. In this work, protein engineering strategy and computer-aided design are performed to evolve the activity and stability of ω-ATA from toward non-natural substrates. After five rounds of mutations, the best variant, M14C3-V5, is obtained, showing better catalytic efficiency toward 1-acetylnaphthalene and higher thermostability than the original enzyme, M14C3. The ro