Our study successfully explores strategies to effectively improve the photocontrol efficiency of light-sensitive enzymes, dubbed photoxenases, with photoswitchable unnatural amino acids (UAAs). The engineering of photoxenases is a versatile method for the reversible photocontrol in various applications. To boost the photocontrol of an established allosteric and heterodimeric photoxenase based on imidazole glycerol phosphate synthase, we turned from an ineffective tuning of the UAA photochemistry to a semi-rational enzyme design. Remarkably, mutations at the catalytically important heterodimer interface increased the light-regulation factor (LRF) for the kcat up to ∼100 with near-quantitative reversibility. Steady-state kinetic investigations combined with computationally determined correlation-based Shortest-Path-Map (SPM) analysis and conformational landscapes revealed how photocontrol was altered in the two best hits. The LRF(kcat) correlated with a shift of a conformational equilibrium between an active and inactive population at the targeted active site and a tuned population productivity upon irradiation. While the overall reduced kcat values originated from a rewiring of the allosteric signal transmission, the increased LRF(kcat) resulted from a change in i) the size of the conformational shift, ii) the population productivity, and iii) the conformational heterogeneity. With this, our findings provide initial guidelines to boost photocontrol and underscore the power of photoxenase engineering.
It has been recently published in Angewandte Chemie International Edition:
Sabrina Mandl, Janet Sánchez, Miquel Estévez, Astrid Bruckmann, Caroline Hiefinger, Sílvia Osuna*, Andrea Hupfeld*
Boosting Reversible Photocontrol of a Photoxenase by an Engineered Conformational Shift
Angew Chem Int Ed, 2026, 65, e18896
DOI: 10.1002/anie.202518896