To develop a selective photoenzyme, we chose the computationally designed Diels-Alderase DA_20_00 as a host scaffold due to mechanistic similarities between [4+2]-and [2+2]-cycloadditions. The DA_20_00 active site contains designed hydrogen bonding residues (Tyr121 and Gln195) intended to promote the Diels-Alder reaction, which may prove useful in supporting catalysis of [2+2]cycloadditions. These residues are embedded within a large hydrophobic pocket suitable for accommodating a bulky aromatic photosensitiser (Figure ). To unlock photocatalytic activity, an engineered Methanococcus jannaschii tyrosyl-tRNAsynthetase/tyrosyl-tRNA (MjTyrRS/MjtRNA Tyr ) pair was used to incorporate 4-benzoylphenylalanine (BpA) at several positions around the DA_20_00 active site pocket (Extended Data Figure ). Intramolecular [2+2]-cycloaddition of quinolone 1 was selected as the target transformation (Figure ). Benzophenone (BP) is a suitable photosensitiser for this reaction and gives rise to racemic straight and crossed chain products (1a and 1b) as a 1.4:1 mixture of regioisomers (Figure ). Placement of BpA at position 173 within the hydrophobic pocket of DA_20_00 provided the active photoenzyme EnT1.0, which is a more effective catalyst than BP and displays modest levels of regio-(2:1, 1a:1b) and enantioselectivity (46% e.e. for the major product (-)-1a). EnT1.0 activity is strictly dependent on light and the presence of the BpA173 photosensitiser (Figure ). . Error bars represent the standard deviation of measurements made in triplicate.