Structure of a switchable subtilisin complexed with a substrate and with the activator azide.
Structure of a switchable subtilisin complexed with a substrate and with the activator azide.
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与底物和激活剂叠氮化物复合的可转换枯草杆菌蛋白酶的结构。
DOI：
10.1021/bi900577n
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发表时间：
2009-11-03
期刊：
影响因子：
2.9
通讯作者：
Bryan, Philip N.
中科院分区：
文献类型：
--
作者：
Gallagher, Travis;Ruan, Biao;London, Mariya;Bryan, Molly A.;Bryan, Philip N.
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 Å 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.
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影响因子：
2.9
作者：
Ruan, B;Fisher, KE;Bryan, PN
通讯作者：
Bryan, PN
影响因子：
2.9
作者：
BRYAN, P;ALEXANDER, P;GALLAGHER, DT
通讯作者：
GALLAGHER, DT
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.9
作者：
Alexander, PA;Ruan, B;Bryan, PN
通讯作者：
Bryan, PN
DOI：
10.1038/nbt0795-669
发表时间：
1995-07-01
期刊：
BIO-TECHNOLOGY
影响因子：
--
作者：
STRAUSBERG, SL;ALEXANDER, PA;BRYAN, PN
通讯作者：
BRYAN, PN