To gain insights into the EnT1.3 catalytic mechanism, a crystal structure of a C-terminally truncated analogue (EnT1.3ΔC310-314, see Supporting Information) complexed with product (-)-1a was solved (1.7 Å, Figure , Supplementary Table ). This truncation has negligible effect on catalytic activity or selectivity (Supplementary Figure ), and was introduced to circumvent the C-terminus of a neighbouring chain from occluding the EnT1.3 active site in crystallo. The ligand sits in a snug active site pocket with its aromatic ring sandwiched between His287 the benzophenone sidechain (3.6 Å and 3.8 Å respectively, Figure , Supplementary Information Figure ). This pose presumably allows for efficient triplet energy transfer from the excited-state photosensitiser to the parent substrate. The crystal structure suggests that Trp244 may play a role in controlling reaction selectivity in favour of the formation of (-)-1a by shaping the active site cavity to prevent addition of the exocyclic alkene to the enantiotopic face of the excited quinolone. Indeed, a W244A mutation leads to a substantial reduction in activity and racemic product formation (Extended Data Figure ). Ligand binding is further supported by complementary hydrogen bonding interactions with Tyr121, which serves as a hydrogen bond acceptor to the quinolone N-H, and Gln195, which acts as a hydrogen bond donor to the quinolone carbonyl (Figure ). Interestingly, Tyr121 and Gln195 were designed to mediate Diels-Alder catalysis in DA_20_00 through hydrogen bonding interactions, albeit with opposite donor/acceptor relationships to those observed in EnT1.3. With substrate 1, mutation of Tyr121 to Phe leads to a 3-fold reduction in activity along with a modest decrease in enantioselectivity (67% e.e., Extended Data Figure ). In contrast, this mutation improves both activity and selectivity towards N-methylated substrate 12 (Figure ), where the N-methyl substituent likely occupies the position vacated by removal of the Tyr121 phenolic oxygen. The hydrogen bond from Gln195 to the quinolone carbonyl is particularly short (2.6 Å) and likely serves to lower the triplet energy of the substrate. Mutation of Gln195 to Ala leads to a considerable reduction in activity and selectivity, underscoring its importance to EnT1.3 catalysis. Gln195 is anchored in position by an additional hydrogen bond with Arg196, which emerged during evolution. Interestingly, in apo-EnT1.3 Gln195 and Arg196 lie in markedly different orientations, with Arg196 instead interacting with Asp149 and Glu225, suggesting that substrate binding may induce formation of a productive Arg196-Gln195-substrate hydrogen bonding network (Extended Data Figure ). This combined structural analysis offers a first glimpse into active site features governing efficient energy transfer catalysis, and provides an important blueprint for the future design of photoenzymes with new and augmented functions. In summary, our study provides a powerful demonstration of how an expanded genetic code can be used to embed entirely new modes of catalysis into proteins. This technology has allowed the development of a proficient photoenzyme that operates via a triplet energy transfer mechanism, versatile reaction manifold in organic synthesis that was previously inaccessible to biocatalysis. While EnT1.3 was tailored to promote photochemical [2+2]-cycloadditions, we anticipate that our approach can be readily adapted to alternative chemistries that are enabled by triplet energy transfer. The catalytic performance and structural sophistication of EnT1.3 is particularly notable, given that <1000 protein variants were evaluated across the evolutionary trajectory. Presumably, more efficient photocatalysts will be generated through a deeper exploration of protein sequence. Likewise, combinations of highthroughput experimentation and in silico design will deliver active sites with new geometries and arrangements of functional groups suitable for mediating selective photocatalysis. Thus with the mechanistic framework for embedding EnT catalysis into proteins now established, we are optimistic about the prospect of developing photoenzymes for a broad array of valuable photochemical processes.