9EZD

BsmI (Bottom Nicking mutant) crystallized with Mg2+ and cognate dsDNA (Post-reactive complex)

9EZD の概要

• エントリーDOI: 10.2210/pdb9ezd/pdb
• 関連するPDBエントリー: 9EZ5 9EZ7
• 分子名称: BsmI, DNA (Bottom strand - 5'-part), DNA (Top strand), ... (8 entities in total)
• 機能のキーワード: restriction endonuclease, dna cleavage, hydrolase
• 由来する生物種: Geobacillus stearothermophilus; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 86343.47

構造登録者

Sieskind, R.,Missoury, S.,Madru, C.,Commenge, I.,Niogret, G.,Rondelez, Y.,Haouz, A.,Legrand, P.,Sauguet, L.,Delarue, M. (登録日: 2024-04-11, 公開日: 2025-04-02)

主引用文献

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

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: 40055548
DOI: 10.1038/s42003-025-07612-z

実験手法

X-RAY DIFFRACTION (2.635 Å)