Biology subjects

Hiefinger, C.

Publications and source records attributed to Hiefinger, C..

3 recordsLinked to original sources

bioRxiv2025

Boosting reversible photocontrol of a photoxenase by an engineered conformational shift

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 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.

biochemistry↗

bioRxiv2025

Expanding the Repertoire of Photoswitchable Unnatural Amino Acids for Enzyme Engineering

Photoswitchable unnatural amino acids (psUAAs) play a crucial role in the engineering of light-sensitivity in enzymes, which holds significant promise for diverse applications such as biotherapy and biocatalysis. Besides near-quantitative photoconversion, the success and expediency of a psUAA for a certain application is defined by its interaction potential with the enzyme, its thermal stability and its effective wavelength of irradiation. To establish high versatility in the current repertoire, we have designed and synthesized six psUAAs based on azobenzene, arylazopyrazole, arylazothiazole, hemithioindigo and spiropyran photoswitches. The resulting psUAAs exhibit an enhanced interaction potential within an enzyme owing to their capacity for hydrogen bonding, ionic interactions, and metal ion coordination. Moreover, we observed diverse photochemical behaviors among the psUAAs, with four of them reversibly switching between the isomers with purely visible light. Notably, we identified orthogonal aminoacyl-tRNA synthetases that facilitate the incorporation of five of the six psUAAs co-translationally and computationally analyzed the synthetase-psUAA interactions. Finally, we evaluated the photochemical behavior of the five psUAAs within an enzymatic model and tested the photocontrol of catalysis confirming their diversity. Ultimately, our findings significantly expanded the repertoire of psUAAs and demonstrated their feasibility for enzyme engineering studies.

synthetic biology↗

bioRxiv2024

Promoting enzyme catalysis via azobenzene facilitated vibrational energy transfer

Azobenzene is a widely recognized tool for achieving artificial spatiotemporal control of enzyme activity using light. Photocontrol reversibility is typically based on photostationary states with varying E and Z isomer compositions achieved through irradiation at specific wavelengths. Here, we report an alternative mechanism for azobenzene based enzyme regulation, discovered through simultaneous irradiation with two wavelengths. Using two engineered variants of imidazole glycerol phosphate synthase, in which azobenzene was incorporated as an unnatural amino acid to enable reversible control under monochromatic irradiation, we uncovered unique behavior under dichromatic irradiation. Notably, a distinct spectroscopic signal from the azobenzene moiety emerged during simultaneous irradiation at 365 nm and 420 nm and vanished upon return to the dark. Intriguingly, dichromatic irradiation triggered a reproducible two-fold increase in catalytic activity and an instantaneous return to baseline activity in the dark for one variant. In contrast, the other variant and the wild-type enzyme maintained their baseline activity under the same conditions. These findings reveal an unexplored avenue for azobenzene photoswitching, offering a novel approach to photocontrol with potential applications in sequentially regulating multiple enzymes, especially when combined with monochromatic irradiation strategies.

biophysics↗