2x2y
From Proteopedia
(Difference between revisions)
(New page: '''Unreleased structure''' The entry 2x2y is ON HOLD Authors: Hekmat, O., Lo Leggio, L., Rosengren, A., Kamarauskaite, J., Kolenova, K., Staalbrand, H. Description: Cellulomonas fimi e...) |
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| - | '''Unreleased structure''' | ||
| - | + | ==Cellulomonas fimi endo-beta-1,4-mannanase double mutant== | |
| + | <StructureSection load='2x2y' size='340' side='right'caption='[[2x2y]], [[Resolution|resolution]] 2.35Å' scene=''> | ||
| + | == Structural highlights == | ||
| + | <table><tr><td colspan='2'>[[2x2y]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Cellulomonas_fimi Cellulomonas fimi]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2X2Y OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2X2Y FirstGlance]. <br> | ||
| + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.35Å</td></tr> | ||
| + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=FMT:FORMIC+ACID'>FMT</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</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=2x2y FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2x2y OCA], [https://pdbe.org/2x2y PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2x2y RCSB], [https://www.ebi.ac.uk/pdbsum/2x2y PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2x2y ProSAT]</span></td></tr> | ||
| + | </table> | ||
| + | == Function == | ||
| + | [https://www.uniprot.org/uniprot/Q9XCV5_CELFI Q9XCV5_CELFI] | ||
| + | == 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/x2/2x2y_consurf.spt"</scriptWhenChecked> | ||
| + | <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> | ||
| + | <text>to colour the structure by Evolutionary Conservation</text> | ||
| + | </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=2x2y ConSurf]. | ||
| + | <div style="clear:both"></div> | ||
| + | <div style="background-color:#fffaf0;"> | ||
| + | == Publication Abstract from PubMed == | ||
| + | To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite -2 and Phe325Ala at subsite -3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce. japonicus enzyme that has an Arg providing mannosyl interactions at subsite -2. The X-ray crystal structure of the C. fimi doubly substituted mannanase was determined to 2.35 A resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite -2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of (18)O-labeled products obtained using H(2)(18)O as a solvent. The results obtained with oligosacharide substrates show that although the catalytic efficiency (k(cat)/K(m)) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite -2 (due to the introduced Arg323) and demotion of it at subsite -3 (to which removal of Phe325 contributed). However, k(cat)/K(m) decreased approximately 1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme. | ||
| - | + | Rational engineering of mannosyl binding in the distal glycone subsites of Cellulomonas fimi endo-beta-1,4-mannanase: mannosyl binding promoted at subsite -2 and demoted at subsite -3 .,Hekmat O, Lo Leggio L, Rosengren A, Kamarauskaite J, Kolenova K, Stalbrand H Biochemistry. 2010 Jun 15;49(23):4884-96. PMID:20426480<ref>PMID:20426480</ref> | |
| - | + | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |
| - | + | </div> | |
| - | + | <div class="pdbe-citations 2x2y" style="background-color:#fffaf0;"></div> | |
| + | == References == | ||
| + | <references/> | ||
| + | __TOC__ | ||
| + | </StructureSection> | ||
| + | [[Category: Cellulomonas fimi]] | ||
| + | [[Category: Large Structures]] | ||
| + | [[Category: Hekmat O]] | ||
| + | [[Category: Kamarauskaite J]] | ||
| + | [[Category: Kolenova K]] | ||
| + | [[Category: Lo Leggio L]] | ||
| + | [[Category: Rosengren A]] | ||
| + | [[Category: Staalbrand H]] | ||
Current revision
Cellulomonas fimi endo-beta-1,4-mannanase double mutant
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