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==E. COLI ALKALINE PHOSPHATASE MUTANT (D153HD330N)== | |||
<StructureSection load='1khk' size='340' side='right'caption='[[1khk]], [[Resolution|resolution]] 2.50Å' scene=''> | |||
| | == Structural highlights == | ||
<table><tr><td colspan='2'>[[1khk]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KHK OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1KHK 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]] 2.5Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=MG:MAGNESIUM+ION'>MG</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=1khk FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1khk OCA], [https://pdbe.org/1khk PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1khk RCSB], [https://www.ebi.ac.uk/pdbsum/1khk PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1khk ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/PPB_ECOLI PPB_ECOLI] | |||
== 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/kh/1khk_consurf.spt"</scriptWhenChecked> | |||
<scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview03.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=1khk ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state. These structures reveal, in particular, that this first mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium fluoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40-fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the first phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (k(cat)), and explains the structural events associated with this artificial evolution. | |||
Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase.,Le Du MH, Lamoure C, Muller BH, Bulgakov OV, Lajeunesse E, Menez A, Boulain JC J Mol Biol. 2002 Mar 1;316(4):941-53. PMID:11884134<ref>PMID:11884134</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 1khk" style="background-color:#fffaf0;"></div> | |||
== | ==See Also== | ||
*[[Alkaline phosphatase 3D structures|Alkaline phosphatase 3D structures]] | |||
== References == | |||
== | <references/> | ||
__TOC__ | |||
</StructureSection> | |||
[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: Boulain | [[Category: Boulain JC]] | ||
[[Category: Bulgakov | [[Category: Bulgakov OV]] | ||
[[Category: Lajeunesse E]] | |||
[[Category: Lajeunesse | [[Category: Lamoure C]] | ||
[[Category: Lamoure | [[Category: Le Du MH]] | ||
[[Category: | [[Category: Menez A]] | ||
[[Category: | [[Category: Muller BH]] | ||
[[Category: | |||
Latest revision as of 07:39, 17 October 2024
E. COLI ALKALINE PHOSPHATASE MUTANT (D153HD330N)E. COLI ALKALINE PHOSPHATASE MUTANT (D153HD330N)
Structural highlights
FunctionEvolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedThe crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state. These structures reveal, in particular, that this first mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium fluoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40-fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the first phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (k(cat)), and explains the structural events associated with this artificial evolution. Artificial evolution of an enzyme active site: structural studies of three highly active mutants of Escherichia coli alkaline phosphatase.,Le Du MH, Lamoure C, Muller BH, Bulgakov OV, Lajeunesse E, Menez A, Boulain JC J Mol Biol. 2002 Mar 1;316(4):941-53. PMID:11884134[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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