# Boosting Reversible Photocontrol of a Photoxenase by an Engineered Conformational Shift

## TL;DR

Researchers improved the light-controlled activity of an enzyme using engineered amino acids and conformational shifts, achieving a 100-fold regulation factor.

## Contribution

A semi-rational enzyme design approach significantly boosted light-regulation efficiency in a heterodimeric photoxenase.

## Key findings

- Mutations at the enzyme's interface increased the light-regulation factor (LRF) for k_cat up to ~100.
- Conformational shifts and population productivity changes were linked to improved photocontrol.
- Allosteric signal transmission rewiring reduced k_cat but enhanced LRF(k_cat).

## Abstract

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 k
cat 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(k
cat) 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 k
cat values originated from a rewiring of the allosteric signal transmission, the increased LRF(k
cat) 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.

Photocontrol of enzymes with photoswitchable unnatural amino acids hitherto yielded only limited light‐regulation factors (LRFs). Here we boosted the LRF of an imidazole glycerol phosphate synthase variant from ∼15 to ∼100 in a semi‐rational design approach and unraveled the mechanistic fundamentals of this effect via biophysical and computational analyses, highlighting the potential and providing guidelines for this photocontrol approach.