Secondary Amine Catalysis in Enzyme Design: Broadening Protein Template Diversity through Genetic Code Expansion

• Published in: Angewandte Chemie International Edition Vol. 63; no. 22; pp. e202403098 - n/a
• Main Authors: Williams, Thomas L., Taily, Irshad M., Hatton, Lewis, Berezin, Andrey A, Wu, Yi‐Lin, Moliner, Vicent, Świderek, Katarzyna, Tsai, Yu‐Hsuan, Luk, Louis Y. P.
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 27.05.2024; John Wiley and Sons Inc
• Edition: International ed. in English
• Subjects: Amines; Artificial Enzyme; Binding; Biocatalysis; Biocatalysts; Biomimetics; Catalysis; Catalytic activity; Dihydrofolate reductase; Enzymes; Fluorescence; Genetic Code; Genetic Code Expansion; Genetic diversity; Green fluorescent protein; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Hydrides; Lysine - analogs & derivatives; Lysine - chemistry; Lysine - metabolism; Nucleophiles; Nucleotides; Organocatalysis; Protein Engineering; Proteins; Proteolysis; Recycling programs; Reductases; Secondary Amine Catalysis; Stereoselectivity; Tetrahydrofolate Dehydrogenase - chemistry; Tetrahydrofolate Dehydrogenase - genetics; Tetrahydrofolate Dehydrogenase - metabolism; Artificial Enzyme; Genetic Code Expansion; Organocatalysis; Secondary Amine Catalysis; Protein Engineering
• DOI: 10.1002/anie.202403098
• ISSN: 1433-7851; 1521-3773

Secondary amines, due to their reactivity, can transform protein templates into catalytically active entities, accelerating the development of artificial enzymes. However, existing methods, predominantly reliant on modified ligands or N‐terminal prolines, impose significant limitations on template selection. In this study, genetic code expansion was used to break this boundary, enabling secondary amines to be incorporated into alternative proteins and positions of choice. Pyrrolysine analogues carrying different secondary amines could be incorporated into superfolder green fluorescent protein (sfGFP), multidrug‐binding LmrR and nucleotide‐binding dihydrofolate reductase (DHFR). Notably, the analogue containing a D‐proline moiety demonstrated both proteolytic stability and catalytic activity, conferring LmrR and DHFR with the desired transfer hydrogenation activity. While the LmrR variants were confined to the biomimetic 1‐benzyl‐1,4‐dihydronicotinamide (BNAH) as the hydride source, the optimal DHFR variant favorably used the pro‐R hydride from NADPH for stereoselective reactions (e.r. up to 92 : 8), highlighting that a switch of protein template could broaden the nucleophile option for catalysis. Owing to the cofactor compatibility, the DHFR‐based secondary amine catalysis could be integrated into an enzymatic recycling scheme. This established method shows substantial potential in enzyme design, applicable from studies on enzyme evolution to the development of new biocatalysts. The importance of protein templates in artificial enzyme design is illustrated through genetic code expansion. Incorporation of a secondary amine into the nucleotide‐binding DHFR and multidrug‐binding LmrR resulted in catalytic entities, with the former favoring the use of NADPH as the hydride source for reactions, whereas the latter required biomimetic 1‐benzyl‐1,4‐dihydronicotinamide (BNAH).