3BGO
Azide complex of Engineered Subtilisin SUBT_BACAM
Summary for 3BGO
| Entry DOI | 10.2210/pdb3bgo/pdb |
| Descriptor | Subtilisin BPN', AZIDE ION, ZINC ION, ... (5 entities in total) |
| Functional Keywords | azide switch, anion sensor, hydrolase, metal-binding, protease, secreted, serine protease, sporulation, zymogen |
| Biological source | Bacillus amyloliquefaciens More |
| Cellular location | Secreted: P00782 P00782 |
| Total number of polymer chains | 2 |
| Total formula weight | 35510.17 |
| Authors | Gallagher, D.T.,Bryan, P.N. (deposition date: 2007-11-27, release date: 2008-06-03, Last modification date: 2024-10-09) |
| Primary citation | 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 Cited by 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: 19761257DOI: 10.1021/bi900577n PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.8 Å) |
Structure validation
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