Reprogramming Stereocontrol Through Progressive Substrate Reorganization in Artificial Triplet Photoenzymes

Precise control of stereoselectivity remains a fundamental challenge in artificial enzymatic catalysis, particularly for artificial photoenzymes that lack evolutionary coupling between photosensitizer positioning and substrate recognition. Herein, by integrating crystallography, kinetics, molecular simulations, and protein engineering, we show that the enantioselectivity of an artificial triplet photoenzyme TPe4.0_FBpA for [2+2] cycloaddition originates from a highly organized indole substrate assembly within the photoactive cavity. Guided by these insights, progressive remodeling of substrate organization through minimal active-site engineering reverses enantiopreference (from 99.5:0.5 e.r. to 2:98 e.r.) while preserving the sequence position of the genetically encoded photosensitizer. Structural and computational analyses further support a switch in the dominant stereocontrol mode from template-assisted substrate organization to dispersion-driven conformational confinement. These findings establish substrate organization as a programmable determinant of stereochemical outcome in artificial photoenzymes and provide a mechanistic framework for stereodivergent excited-state biocatalysis.