Molecular plasticity in the flavin binding pocket of BLUF domain evolves first light-gated endonuclease in bacterial system
The bacterium Rubellimicrobium mesophilum possesses a 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 evolutionarily replaced by phenylalanine (Y5F), asparagine (H27N) and alanine (W87A) residues, respectively. In present communication, we have studied the impact of this evolutionary plasticity on the BLUF photodynamics and associated endonuclease activity. The results obtained showed that the evolutionary plasticity in the BLUF domain influenced various functional aspects of BLUF domain including FAD binding, domain stability, recovery kinetics, and spectral characteristics. The impact of amino acid plasticity on the C-terminal endonuclease (EndoIII) domain was also studied. The evolutionary plasticity induced changes in the flavin binding pocket of the BLUF domain elevated the light-gated endonuclease activity associated with EndoIII domain. The molecular docking analysis and spectroscopic studies also confirmed the substrate-binding ability of the BLUF-EndoIII. The elevated endonuclease activity suggested that the amino acid residues, which are crucial for BLUF photocycle are indeed dispensable and there might exists another electron transfer pathway for BLUF domain activation and regulation of associated endonuclease domain. Considering the role of endonucleases in bacterial defense, the understanding of the BLUF photodynamics, mechanism of signal transfer to the downstream endonuclease domain and associated endonuclease activity might elucidate the first naturally occurring light-gated endonuclease in bacterial system.