Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands

Commun Biol. 2025 Mar 7;8(1):387. doi: 10.1038/s42003-025-07612-z.

Abstract

BsmI, a thermophilic Type IIS restriction endonuclease from Bacillus stearothermophilus, presents a unique structural composition, housing two distinct active sites within a single monomer. Recognition of the non-symmetrical 5'-GAATGC-3' sequence enables precise cleavage of the top and bottom DNA strands. Synthetic biology interventions have led to the transformation of BsmI into Nb.BsmI, a nicking endonuclease. Here we introduce Nt*.BsmI, tailored for top-strand cleavage, which is inactive on standard double-stranded DNA, but active on bottom-strand nicked DNA, suggesting a sequential cleavage mechanism. Crystallographic structures of pre- and post-reactive complexes with cognate DNA show one major conformational change, a retractable loop possibly governing sequential active site accessibility. The x-ray structures reveal the position of the divalent metal ions in the active sites and the DNA:protein interactions, while the models predicted by Alphafold3 are incorrect. This comprehensive structural and functional study lays a foundation for rational enzyme redesign and potential applications in biotechnology.

MeSH terms

  • Bacterial Proteins* / chemistry
  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / metabolism
  • Catalytic Domain
  • Crystallography, X-Ray
  • DNA Cleavage*
  • DNA* / chemistry
  • DNA* / metabolism
  • Deoxyribonucleases, Type II Site-Specific* / chemistry
  • Deoxyribonucleases, Type II Site-Specific* / genetics
  • Deoxyribonucleases, Type II Site-Specific* / metabolism
  • Geobacillus stearothermophilus* / enzymology
  • Geobacillus stearothermophilus* / genetics
  • Models, Molecular
  • Protein Conformation

Substances

  • Deoxyribonucleases, Type II Site-Specific
  • DNA
  • Bacterial Proteins