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3d2a

From Proteopedia

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Structure of 1-17A4, a thermostable mutant of Bacillus subtilis lipase obtained through directed evolution

Structural highlights

3d2a is a 1 chain structure with sequence from Bacillus subtilis. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.73Å
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

ESTA_BACSU Active toward p-nitrophenyl esters and triacylglycerides with a marked preference for esters with C8 acyl groups.^[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 PubMed

In vitro evolution methods are now being routinely used to identify protein variants with novel and enhanced properties that are difficult to achieve using rational design. However, one of the limitations is in screening for beneficial mutants through several generations due to the occurrence of neutral/negative mutations occurring in the background of positive ones. While evolving a lipase in vitro from mesophilic Bacillus subtilis to generate thermostable variants, we have designed protocols that combine stringent three-tier testing, sequencing and stability assessments on the protein at the end of each generation. This strategy resulted in a total of six stabilizing mutations in just two generations with three mutations per generation. Each of the six mutants when evaluated individually contributed additively to thermostability. A combination of all of them resulted in the best variant that shows a remarkable 15 degrees C shift in melting temperature and a millionfold decrease in the thermal inactivation rate with only a marginal increase of 3 kcal mol(-1) in free energy of stabilization. Notably, in addition to the dramatic shift in optimum temperature by 20 degrees C, the activity has increased two- to fivefold in the temperature range 25-65 degrees C. High-resolution crystal structures of three of the mutants, each with 5 degrees increments in melting temperature, reveal the structural basis of these mutations in attaining higher thermostability. The structures highlight the importance of water-mediated ionic networks on the protein surface in imparting thermostability. Saturation mutagenesis at each of the six positions did not result in enhanced thermostability in almost all the cases, confirming the crucial role played by each mutation as revealed through the structural study. Overall, our study presents an efficient strategy that can be employed in directed evolution approaches employed for obtaining improved properties of proteins.

Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight.,Ahmad S, Kamal MZ, Sankaranarayanan R, Rao NM J Mol Biol. 2008 Aug 29;381(2):324-40. Epub 2008 Jul 2. PMID:18599073^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Lesuisse E, Schanck K, Colson C. Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme. Eur J Biochem. 1993 Aug 15;216(1):155-60. PMID:8396026
↑ Ahmad S, Kamal MZ, Sankaranarayanan R, Rao NM. Thermostable Bacillus subtilis lipases: in vitro evolution and structural insight. J Mol Biol. 2008 Aug 29;381(2):324-40. Epub 2008 Jul 2. PMID:18599073 doi:10.1016/j.jmb.2008.05.063

1 Structural highlights
2 Function
3 Evolutionary Conservation
4 Publication Abstract from PubMed
5 See Also
6 References

Proteopedia Page Contributors and Editors (what is this?)

OCA

Retrieved from "http://52.214.119.220/wiki/index.php/3d2a"

Categories: Bacillus subtilis | Large Structures | Kamal MZ | Sankaranarayanan R

@@ Line 1: / Line 1: @@
 ==Structure of 1-17A4, a thermostable mutant of Bacillus subtilis lipase obtained through directed evolution==
-<StructureSection load='3d2a' size='340' side='right' caption='[[3d2a]], [[Resolution|resolution]] 1.73&Aring;' scene=''>
+<StructureSection load='3d2a' size='340' side='right'caption='[[3d2a]], [[Resolution|resolution]] 1.73&Aring;' scene=''>
 == Structural highlights ==
-<table><tr><td colspan='2'>[[3d2a]] is a 1 chain structure with sequence from [http://en.wikipedia.org/wiki/"bacillus_globigii"_migula_1900 "bacillus globigii" migula 1900]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3D2A OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3D2A FirstGlance]. <br>
+<table><tr><td colspan='2'>[[3d2a]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Bacillus_subtilis Bacillus subtilis]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3D2A OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3D2A FirstGlance]. <br>
-</td></tr><tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[1t2n|1t2n]], [[1t4m|1t4m]], [[3d2b|3d2b]], [[3d2c|3d2c]]</td></tr>
+</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.73&#8491;</td></tr>
-<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">lipA, lip, BSU02700 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=1423 "Bacillus globigii" Migula 1900])</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=3d2a FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3d2a OCA], [https://pdbe.org/3d2a PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3d2a RCSB], [https://www.ebi.ac.uk/pdbsum/3d2a PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3d2a ProSAT]</span></td></tr>
-<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Triacylglycerol_lipase Triacylglycerol lipase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.1.3 3.1.1.3] </span></td></tr>
-<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3d2a FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3d2a OCA], [http://pdbe.org/3d2a PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=3d2a RCSB], [http://www.ebi.ac.uk/pdbsum/3d2a PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=3d2a ProSAT]</span></td></tr>
 </table>
 == Function ==
-[[http://www.uniprot.org/uniprot/ESTA_BACSU ESTA_BACSU]] Active toward p-nitrophenyl esters and triacylglycerides with a marked preference for esters with C8 acyl groups.<ref>PMID:8396026</ref>
+[https://www.uniprot.org/uniprot/ESTA_BACSU ESTA_BACSU] Active toward p-nitrophenyl esters and triacylglycerides with a marked preference for esters with C8 acyl groups.<ref>PMID:8396026</ref>
 == Evolutionary Conservation ==
 [[Image:Consurf_key_small.gif|200px|right]]
 Check<jmol>
   <jmolCheckbox>
-    <scriptWhenChecked>select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/d2/3d2a_consurf.spt"</scriptWhenChecked>
+    <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/d2/3d2a_consurf.spt"</scriptWhenChecked>
     <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked>
     <text>to colour the structure by Evolutionary Conservation</text>
@@ Line 30: / Line 28: @@
 </div>
 <div class="pdbe-citations 3d2a" style="background-color:#fffaf0;"></div>
+==See Also==
+*[[Lipase 3D Structures|Lipase 3D Structures]]
 == References ==
 <references/>
 __TOC__
 </StructureSection>
-[[Category: Bacillus globigii migula 1900]]
+[[Category: Bacillus subtilis]]
-[[Category: Triacylglycerol lipase]]
+[[Category: Large Structures]]
-[[Category: Kamal, M Z]]
+[[Category: Kamal MZ]]
-[[Category: Sankaranarayanan, R]]
+[[Category: Sankaranarayanan R]]
-[[Category: Alpha/beta hydrolase]]
-[[Category: Directed evolution]]
-[[Category: Hydrolase]]
-[[Category: Lipase]]
-[[Category: Lipid degradation]]
-[[Category: Secreted]]
-[[Category: Stability]]

3d2a

From Proteopedia

Current revision

Structure of 1-17A4, a thermostable mutant of Bacillus subtilis lipase obtained through directed evolution

Structural highlights

Function

Evolutionary Conservation

Publication Abstract from PubMed

See Also

References

Contents

Proteopedia Page Contributors and Editors (what is this?)

Views

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