9ENO

Crystal structure of ComplemEnT1.4 (CEnT1.4), an engineered photoenzyme for selective [2+2]-cycloadditions

Summary for 9ENO

• Entry DOI: 10.2210/pdb9eno/pdb
• Descriptor: Diisopropyl-fluorophosphatase, 1,2-ETHANEDIOL, DI(HYDROXYETHYL)ETHER, ... (5 entities in total)
• Functional Keywords: photoenzyme, [2+2]-cyclase, artificial enzyme, biosynthetic protein
• Biological source: Loligo vulgaris
• Total number of polymer chains: 2
• Total formula weight: 73657.54

Authors

Hardy, F.J.,Crawshaw, R.C.,Green, A.P. (deposition date: 2024-03-13, release date: 2025-07-16, Last modification date: 2025-09-03)

Primary citation

Sun, C.,Kohn, A.R.,Smithson, R.,Hardy, F.J.,Trimble, J.S.,Cao, Y.,Johannissen, L.O.,Hay, S.,Crawshaw, R.,Green, A.P.
Photosensitizer Repositioning Affords an Enantiocomplementary Enzyme for [2 + 2]-Cycloadditions.
Angew.Chem.Int.Ed.Engl., 64:e202503576-e202503576, 2025

PubMed Abstract: The combination of genetic code expansion and directed evolution has recently given rise to enantioselective photoenzymes for [2 + 2]-cycloadditions of quinolone and indole derivatives. However, the enzymes reported to date only allow access to one enantiomeric series of the strained cyclobutane products. Here, guided by a crystal structure of our previously engineered enzyme EnT1.3, we show how judicious repositioning of the genetically programmed benzophenone photosensitizer affords an enantiocomplementary [2 + 2]-cyclase, CEnT1.0. Following directed evolution, a proficient and oxygen-tolerant photoenzyme (CEnT1.4) emerged that promotes [2 + 2]-cycloadditions of a quinolone derivative with exquisite enantiocontrol (99% e.e.) and substantially enhanced regioselectivity compared with EnT1.3 (r.r. 62:1 vs. 9:1). Structural analysis of CEnT1.4, coupled with molecular dynamic simulations, reveals a well-sculpted active site pocket that pre-organises the substrate for regio- and enantioselective catalysis. This study highlights the versatility offered by genetically programmed (photo)catalytic elements when developing enzymes for altered stereochemical outcomes.

PubMed: 40576080
DOI: 10.1002/anie.202503576

Experimental method

X-RAY DIFFRACTION (1.73 Å)