9EZD
BsmI (Bottom Nicking mutant) crystallized with Mg2+ and cognate dsDNA (Post-reactive complex)
Summary for 9EZD
| Entry DOI | 10.2210/pdb9ezd/pdb |
| Related | 9EZ5 9EZ7 |
| Descriptor | BsmI, DNA (Bottom strand - 5'-part), DNA (Top strand), ... (8 entities in total) |
| Functional Keywords | restriction endonuclease, dna cleavage, hydrolase |
| Biological source | Geobacillus stearothermophilus More |
| Total number of polymer chains | 4 |
| Total formula weight | 86343.47 |
| Authors | Sieskind, R.,Missoury, S.,Madru, C.,Commenge, I.,Niogret, G.,Rondelez, Y.,Haouz, A.,Legrand, P.,Sauguet, L.,Delarue, M. (deposition date: 2024-04-11, release date: 2025-04-02) |
| Primary citation | Sieskind, R.,Missoury, S.,Madru, C.,Commenge, I.,Niogret, G.,Hollenstein, M.,Rondelez, Y.,Sauguet, L.,Haouz, A.,Legrand, P.,Delarue, M. Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands. Commun Biol, 8:387-387, 2025 Cited by PubMed 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. PubMed: 40055548DOI: 10.1038/s42003-025-07612-z PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (2.635 Å) |
Structure validation
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