3BGO

Azide complex of Engineered Subtilisin SUBT_BACAM

3BGO の概要

• エントリーDOI: 10.2210/pdb3bgo/pdb
• 分子名称: Subtilisin BPN', AZIDE ION, ZINC ION, ... (5 entities in total)
• 機能のキーワード: azide switch, anion sensor, hydrolase, metal-binding, protease, secreted, serine protease, sporulation, zymogen
• 由来する生物種: Bacillus amyloliquefaciens; 詳細
• 細胞内の位置: Secreted: P00782 P00782
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 35510.17

構造登録者

Gallagher, D.T.,Bryan, P.N. (登録日: 2007-11-27, 公開日: 2008-06-03, 最終更新日: 2024-10-09)

主引用文献

Gallagher, T.,Ruan, B.,London, M.,Bryan, M.A.,Bryan, P.N.
Structure of a switchable subtilisin complexed with a substrate and with the activator azide.
Biochemistry, 48:10389-10394, 2009

PubMed Abstract: An engineered variant of the protease subtilisin from Bacillus amyloliquefaciens, in which the D32A mutation renders the enzyme's activity dependent on the presence of certain small anions such as fluoride or azide, has been produced. This modified enzyme has applications as an azide or fluoride-triggered expression-purification tool. We report activity measurements showing that the enzyme is activated more than 3000-fold by azide and describe the 1.8 A resolution structure of an inactive form (by replacing the catalytic nucleophile Ser 221 with alanine) of the protease, in complex with azide and with a substrate that spans the active site. Both enzyme and substrate have been engineered to increase their stability and the affinity of their interaction. The substrate is based on a stabilized subtilisin prodomain, extended across the active site by the addition of four residues at its C-terminus. In the crystal structure, the substrate is well-ordered across the active site, and the azide anion is observed bound adjacent to Ala 32. The structures of the substrate complex in three different crystals (anion-free, fluoride-soaked, and azide-soaked) are compared. These structures provide extensive information for understanding subtilisin's substrate binding and catalytic mechanism, and for the development of biotechnology tools based on anion-activated proteolysis. The mechanism of anion-dependent proteolysis appears to be a slight modification of the accepted charge-relay mechanism for serine proteases.

PubMed: 19761257
DOI: 10.1021/bi900577n

実験手法

X-RAY DIFFRACTION (1.8 Å)