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[[ | ==Hypocrea jecorina Cel6A D221A mutant soaked with 6-chloro-4- methylumbelliferyl-belta-D-cellobioside== | ||
<StructureSection load='4au0' size='340' side='right' caption='[[4au0]], [[Resolution|resolution]] 1.70Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[4au0]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Trichoderma_reesei Trichoderma reesei]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4AU0 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4AU0 FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=BGC:BETA-D-GLUCOSE'>BGC</scene>, <scene name='pdbligand=MAN:ALPHA-D-MANNOSE'>MAN</scene>, <scene name='pdbligand=NAG:N-ACETYL-D-GLUCOSAMINE'>NAG</scene>, <scene name='pdbligand=XZZ:6-CHLORO-7-HYDROXY-4-METHYL-2H-CHROMEN-2-ONE'>XZZ</scene></td></tr> | |||
<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[1cb2|1cb2]], [[1hgw|1hgw]], [[1hgy|1hgy]], [[1qjw|1qjw]], [[1qk0|1qk0]], [[1qk2|1qk2]], [[3cbh|3cbh]], [[4ax6|4ax6]], [[4ax7|4ax7]]</td></tr> | |||
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Cellulose_1,4-beta-cellobiosidase Cellulose 1,4-beta-cellobiosidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.91 3.2.1.91] </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=4au0 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4au0 OCA], [http://www.rcsb.org/pdb/explore.do?structureId=4au0 RCSB], [http://www.ebi.ac.uk/pdbsum/4au0 PDBsum]</span></td></tr> | |||
</table> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Methylumbelliferyl-beta-cellobioside (MUF-G2) is a convenient fluorogenic substrate for certain beta-glycoside hydrolases (GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes (GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF-G2 in the +1 subsite of Hypocrea jecorina Cel6A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-beta-cellobiosides [6-chloro-4-methyl- (ClMUF); 6-chloro-4-trifluoromethyl- (ClF3MUF); 4-phenyl- (PhUF); 6-chloro-4-phenyl- (ClPhUF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6A was 10-150 times higher than with MUF-G2, although it was still three orders of magnitude lower than with H. jecorina Cel7B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while ClMUF-G2 could be used for determination of k(cat) and K(M) for H. jecorina Cel6A and Thermobifida fusca Cel6A. Crystal structures of H. jecorina Cel6A D221A mutant soaked with the MUF-, ClMUF- and ClPhUF-beta-cellobioside substrates show that the modifications turned the umbelliferyl group 'upside down', with the glycosidic bond better positioned for protonation than with MUF-G2. DATABASE: Structural data have been submitted to the Protein Data Bank under accession numbers pdb 4AU0, 4AX7, 4AX6 STRUCTURED DIGITAL ABSTRACT: * http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260296 * Cel6A and Cel6A bind by x-ray crystallography (View Interaction: 1, 2). | |||
Rational design, synthesis, evaluation and enzyme-substrate structures of improved fluorogenic substrates for family 6 glycoside hydrolases.,Wu M, Nerinckx W, Piens K, Ishida T, Hansson H, Sandgren M, Stahlberg J FEBS J. 2013 Jan;280(1):184-98. doi: 10.1111/febs.12060. Epub 2012 Dec 7. PMID:23137336<ref>PMID:23137336</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
== References == | |||
<references/> | |||
== | __TOC__ | ||
</StructureSection> | |||
[[Category: Cellulose 1,4-beta-cellobiosidase]] | [[Category: Cellulose 1,4-beta-cellobiosidase]] | ||
[[Category: Trichoderma reesei]] | [[Category: Trichoderma reesei]] | ||
[[Category: Hansson, H | [[Category: Hansson, H]] | ||
[[Category: Ishida, T | [[Category: Ishida, T]] | ||
[[Category: Nerinckx, W | [[Category: Nerinckx, W]] | ||
[[Category: Piens, K | [[Category: Piens, K]] | ||
[[Category: Sandgren, M | [[Category: Sandgren, M]] | ||
[[Category: Stahlberg, J | [[Category: Stahlberg, J]] | ||
[[Category: Wu, M | [[Category: Wu, M]] | ||
[[Category: Cellulase]] | [[Category: Cellulase]] | ||
[[Category: Clmufg2]] | [[Category: Clmufg2]] | ||
Revision as of 17:39, 9 December 2014
Hypocrea jecorina Cel6A D221A mutant soaked with 6-chloro-4- methylumbelliferyl-belta-D-cellobiosideHypocrea jecorina Cel6A D221A mutant soaked with 6-chloro-4- methylumbelliferyl-belta-D-cellobioside
Structural highlights
Publication Abstract from PubMedMethylumbelliferyl-beta-cellobioside (MUF-G2) is a convenient fluorogenic substrate for certain beta-glycoside hydrolases (GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes (GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF-G2 in the +1 subsite of Hypocrea jecorina Cel6A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-beta-cellobiosides [6-chloro-4-methyl- (ClMUF); 6-chloro-4-trifluoromethyl- (ClF3MUF); 4-phenyl- (PhUF); 6-chloro-4-phenyl- (ClPhUF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6A was 10-150 times higher than with MUF-G2, although it was still three orders of magnitude lower than with H. jecorina Cel7B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while ClMUF-G2 could be used for determination of k(cat) and K(M) for H. jecorina Cel6A and Thermobifida fusca Cel6A. Crystal structures of H. jecorina Cel6A D221A mutant soaked with the MUF-, ClMUF- and ClPhUF-beta-cellobioside substrates show that the modifications turned the umbelliferyl group 'upside down', with the glycosidic bond better positioned for protonation than with MUF-G2. DATABASE: Structural data have been submitted to the Protein Data Bank under accession numbers pdb 4AU0, 4AX7, 4AX6 STRUCTURED DIGITAL ABSTRACT: * http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260296 * Cel6A and Cel6A bind by x-ray crystallography (View Interaction: 1, 2). Rational design, synthesis, evaluation and enzyme-substrate structures of improved fluorogenic substrates for family 6 glycoside hydrolases.,Wu M, Nerinckx W, Piens K, Ishida T, Hansson H, Sandgren M, Stahlberg J FEBS J. 2013 Jan;280(1):184-98. doi: 10.1111/febs.12060. Epub 2012 Dec 7. PMID:23137336[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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