Secondary amine catalysis in enzyme design: broadening protein template diversity through genetic code expansion

Williams, Thomas L. ORCID: https://orcid.org/0000-0003-1710-3914, Taily, Irshad M., Hatton, Lewis, Berezin, Andrey A, Wu, Yi‐Lin ORCID: https://orcid.org/0000-0003-0253-1625, Moliner, Vicent, Świderek, Katarzyna, Tsai, Yu‐Hsuan ORCID: https://orcid.org/0000-0003-0589-5088 and Luk, Louis Y. P. ORCID: https://orcid.org/0000-0002-7864-6261 2024. Secondary amine catalysis in enzyme design: broadening protein template diversity through genetic code expansion. Angewandte Chemie International Edition 10.1002/anie.202403098

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Abstract

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.

Item Type:	Article
Date Type:	Published Online
Status:	Published
Schools:	Chemistry Cardiff Catalysis Institute (CCI)
Additional Information:	License information from Publisher: LICENSE 1: URL: http://creativecommons.org/licenses/by/4.0/
Publisher:	Wiley
ISSN:	1433-7851
Funders:	EPSRC, BBSRC, Royal Society
Date of First Compliant Deposit:	22 April 2024
Date of Acceptance:	27 March 2024
Last Modified:	29 Apr 2024 14:02
URI:	https://orca.cardiff.ac.uk/id/eprint/168212

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