Photo-Regulation of Enzyme Activity: The Inactivation of a Carboligase with Genetically Encoded Photosensitizer Fusion Tags

Genetically encoded photosensitizers are able to produce reactive oxygen species upon illumination and exploited in a wide range of applications, especially the medical field. In this work, we envisioned further apply these genetically for light-dependent control single enzymes multi-step biocatalysis. One challenges application several cascade is unwanted cross-reactivity biocatalysts on reaction intermediates when all simultaneously present reaction. As one strategy address issue, investigated whether introduction as fusion tags would allow selective inactivation after successful transformation by simply turning light. We tested five different molecular biological inactivate pyruvate decarboxylase variant E469G/W543H from Acetobacter pasteurianus . Dimeric photosensitizer tags, like flavin-binding fluorescent proteins Bacillus subtilis Pseudomonas putida showed tendency form insoluble protein aggregates combination with tetrameric carboligase. Enzyme activity was, some extent, retained aggregates, but handling proved be unfeasible. Monomeric appeared much more suitable fused enzyme. dark, singlet photosensitizing (SOPP3)-tagged carboligase 79% its compared unfused Upon blue light exposure, SOPP3 tag best specific enabled complete within 30 min. thus seen promising applied future enzyme cascades overcome challenge cross-reactivity.