Mandl, Sabrina and Sanchez, Janet and Estevez, Miquel and Bruckmann, Astrid and Hiefinger, Caroline and Osuna, Silvia and Hupfeld, Andrea (2026) Boosting Reversible Photocontrol of a Photoxenase by an Engineered Conformational Shift. ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 65 (4). ISSN 1521-3773
Full text not available from this repository. (Request a copy)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 similar 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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | GLYCEROL PHOSPHATE SYNTHASE; AMINO-ACID; GLUTAMINE AMIDOTRANSFERASE; ALLOSTERIC ACTIVATION; MILLISECOND MOTIONS; CODON REASSIGNMENT; ATOMIC CHARGES; PROTEIN; MECHANISM; BIENZYME; Correlation-based methods; Enzyme catalysis; Molecular dynamics; Photocontrol; Unnatural amino acids |
| Subjects: | 500 Science > 540 Chemistry & allied sciences 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 23 Sep 2026 04:21 |
| Last Modified: | 23 Sep 2026 04:21 |
| URI: | https://pred.uni-regensburg.de/id/eprint/66395 |
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