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==Azide complex of Engineered Subtilisin SUBT_BACAM== | ==Azide complex of Engineered Subtilisin SUBT_BACAM== | ||
<StructureSection load='3bgo' size='340' side='right' caption='[[3bgo]], [[Resolution|resolution]] 1.80Å' scene=''> | <StructureSection load='3bgo' size='340' side='right'caption='[[3bgo]], [[Resolution|resolution]] 1.80Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>[[3bgo]] is a 2 chain structure with sequence from [ | <table><tr><td colspan='2'>[[3bgo]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Bacillus_amyloliquefaciens Bacillus amyloliquefaciens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3BGO OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3BGO FirstGlance]. <br> | ||
</td></tr><tr id=' | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.8Å</td></tr> | ||
<tr id=' | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=AZI:AZIDE+ION'>AZI</scene>, <scene name='pdbligand=ZN:ZINC+ION'>ZN</scene></td></tr> | ||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=3bgo FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3bgo OCA], [https://pdbe.org/3bgo PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3bgo RCSB], [https://www.ebi.ac.uk/pdbsum/3bgo PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3bgo ProSAT]</span></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[ | |||
</table> | </table> | ||
== Function == | == Function == | ||
[ | [https://www.uniprot.org/uniprot/SUBT_BACAM SUBT_BACAM] Subtilisin is an extracellular alkaline serine protease, it catalyzes the hydrolysis of proteins and peptide amides. Has a high substrate specificity to fibrin.<ref>PMID:12524032</ref> | ||
== Evolutionary Conservation == | == Evolutionary Conservation == | ||
[[Image:Consurf_key_small.gif|200px|right]] | [[Image:Consurf_key_small.gif|200px|right]] | ||
Check<jmol> | Check<jmol> | ||
<jmolCheckbox> | <jmolCheckbox> | ||
<scriptWhenChecked>select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/bg/3bgo_consurf.spt"</scriptWhenChecked> | <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/bg/3bgo_consurf.spt"</scriptWhenChecked> | ||
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/ | <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview03.spt</scriptWhenUnchecked> | ||
<text>to colour the structure by Evolutionary Conservation</text> | <text>to colour the structure by Evolutionary Conservation</text> | ||
</jmolCheckbox> | </jmolCheckbox> | ||
</jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=3bgo ConSurf]. | </jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=3bgo ConSurf]. | ||
<div style="clear:both"></div> | <div style="clear:both"></div> | ||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
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. | |||
Structure of a switchable subtilisin complexed with a substrate and with the activator azide.,Gallagher T, Ruan B, London M, Bryan MA, Bryan PN Biochemistry. 2009 Nov 3;48(43):10389-94. doi: 10.1021/bi900577n. PMID:19761257<ref>PMID:19761257</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 3bgo" style="background-color:#fffaf0;"></div> | |||
==See Also== | ==See Also== | ||
*[[Subtilisin|Subtilisin]] | *[[Subtilisin 3D structures|Subtilisin 3D structures]] | ||
== References == | == References == | ||
<references/> | <references/> | ||
| Line 28: | Line 37: | ||
</StructureSection> | </StructureSection> | ||
[[Category: Bacillus amyloliquefaciens]] | [[Category: Bacillus amyloliquefaciens]] | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: Bryan | [[Category: Bryan PN]] | ||
[[Category: Gallagher | [[Category: Gallagher DT]] | ||
Latest revision as of 10:53, 9 October 2024
Azide complex of Engineered Subtilisin SUBT_BACAMAzide complex of Engineered Subtilisin SUBT_BACAM
Structural highlights
FunctionSUBT_BACAM Subtilisin is an extracellular alkaline serine protease, it catalyzes the hydrolysis of proteins and peptide amides. Has a high substrate specificity to fibrin.[1] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedAn 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. Structure of a switchable subtilisin complexed with a substrate and with the activator azide.,Gallagher T, Ruan B, London M, Bryan MA, Bryan PN Biochemistry. 2009 Nov 3;48(43):10389-94. doi: 10.1021/bi900577n. PMID:19761257[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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