Molecular plasticity in the flavin-binding pocket of a BLUF domain enables an efficient light-gated endonuclease.

The bacterium Rubellimicrobium mesophilum possesses a blue light-using flavin (BLUF)-coupled endonuclease III (BLUF-EndoIII) with potential endonuclease activity. Interestingly, the crucial amino acid residues (tyrosine, histidine, and tryptophan) responsible for BLUF photocycle and photodynamics are replaced by phenylalanine (Y5F), asparagine (H27N), and alanine (W87A) residues, respectively. In the present study, we investigated the impact of this evolutionary plasticity on BLUF photodynamics and the associated endonuclease activity. The results obtained showed that the residue exchanges within the BLUF domain influenced its functional aspects, including flavin binding, domain stability, recovery kinetics, and spectral characteristics. The evolutionary plasticity-induced changes in the flavin-binding pocket of the BLUF domain elevated the light-gated endonuclease activity associated with the EndoIII domain. Molecular docking analysis and spectroscopic studies confirmed the substrate-binding ability of BLUF-EndoIII. Elevated endonuclease activity suggested that the amino acid residues, previously deemed crucial for the BLUF photocycle, are indeed dispensable, and there might exist another pathway for BLUF domain activation and regulation of the associated endonuclease domain. Considering the role of endonucleases in bacterial defense, it is crucial to understand the BLUF photodynamics and mechanism of signal transfer to the downstream endonuclease domain. This understanding elucidates the functioning of the naturally occurring light-gated endonuclease BLUF-EndoIII in the bacterium Rubellimicrobium mesophilum.