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New page: left|200px<br /><applet load="2d0d" size="350" color="white" frame="true" align="right" spinBox="true" caption="2d0d, resolution 1.65Å" /> '''Crystal Structure of... |
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== | ==Crystal Structure of a Meta-cleavage Product Hydrolase (CumD) A129V Mutant== | ||
The meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 | <StructureSection load='2d0d' size='340' side='right'caption='[[2d0d]], [[Resolution|resolution]] 1.65Å' scene=''> | ||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[2d0d]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Pseudomonas_fluorescens Pseudomonas fluorescens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2D0D OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2D0D 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]] 1.65Å</td></tr> | |||
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=PO4:PHOSPHATE+ION'>PO4</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=2d0d FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2d0d OCA], [https://pdbe.org/2d0d PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2d0d RCSB], [https://www.ebi.ac.uk/pdbsum/2d0d PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2d0d ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/P96965_PSEFL P96965_PSEFL] | |||
== 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/d0/2d0d_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=2d0d ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
The meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) hydrolyzes 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl HODA) in the cumene (isopropylbenzene) degradation pathway. To modulate the substrate specificity and catalytic efficiency of CumD toward substrates derived from monocyclic aromatic compounds, we constructed the CumD mutants, A129V, I199V, and V227I, as well as four types of double and triple mutants. Toward substrates with smaller side chains (e.g. 2-hydroxy-6-oxohepta-2,4-dienoate; 6-ethyl-HODA), the k(cat)/K(m) values of the single mutants were 4.2-11 fold higher than that of the wild type enzyme and 1.8-4.7 fold higher than that of the meta-cleavage product hydrolase from Pseudomonas putida F1 (TodF). The A129V mutant showed the highest k(cat)/K(m) value for 2-hydroxy-6-oxohepta-2,4-dienoate (6-ethyl-HODA). The crystal structure of the A129V mutant was determined at 1.65 A resolution, enabling location of the Ogamma atom of the Ser103 side chain. A chloride ion was bound to the oxyanion hole of the active site, and mutant enzymes at the residues forming this site were also examined. The k(cat) values of Ser34 mutants were decreased 2.9-65 fold, suggesting that the side chain of Ser34 supports catalysis by stabilizing the anionic oxygen of the proposed intermediate state (gem-diolate). This is the first crystal structure determination of CumD in an active form, with the Ser103 residue, one of the catalytically essential "triad", being intact. | |||
Improving the catalytic efficiency of a meta-cleavage product hydrolase (CumD) from Pseudomonas fluorescens IP01.,Jun SY, Fushinobu S, Nojiri H, Omori T, Shoun H, Wakagi T Biochim Biophys Acta. 2006 Jul;1764(7):1159-66. Epub 2006 Jun 7. PMID:16844437<ref>PMID:16844437</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
[[Category: | <div class="pdbe-citations 2d0d" style="background-color:#fffaf0;"></div> | ||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Large Structures]] | |||
[[Category: Pseudomonas fluorescens]] | [[Category: Pseudomonas fluorescens]] | ||
[[Category: Fushinobu S]] | |||
[[Category: Fushinobu | [[Category: Jun SY]] | ||
[[Category: Jun | [[Category: Nojiri H]] | ||
[[Category: Nojiri | [[Category: Omori T]] | ||
[[Category: Omori | [[Category: Shoun H]] | ||
[[Category: Shoun | [[Category: Wakagi T]] | ||
[[Category: Wakagi | |||
Latest revision as of 11:21, 25 October 2023
Crystal Structure of a Meta-cleavage Product Hydrolase (CumD) A129V MutantCrystal Structure of a Meta-cleavage Product Hydrolase (CumD) A129V Mutant
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 meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) hydrolyzes 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl HODA) in the cumene (isopropylbenzene) degradation pathway. To modulate the substrate specificity and catalytic efficiency of CumD toward substrates derived from monocyclic aromatic compounds, we constructed the CumD mutants, A129V, I199V, and V227I, as well as four types of double and triple mutants. Toward substrates with smaller side chains (e.g. 2-hydroxy-6-oxohepta-2,4-dienoate; 6-ethyl-HODA), the k(cat)/K(m) values of the single mutants were 4.2-11 fold higher than that of the wild type enzyme and 1.8-4.7 fold higher than that of the meta-cleavage product hydrolase from Pseudomonas putida F1 (TodF). The A129V mutant showed the highest k(cat)/K(m) value for 2-hydroxy-6-oxohepta-2,4-dienoate (6-ethyl-HODA). The crystal structure of the A129V mutant was determined at 1.65 A resolution, enabling location of the Ogamma atom of the Ser103 side chain. A chloride ion was bound to the oxyanion hole of the active site, and mutant enzymes at the residues forming this site were also examined. The k(cat) values of Ser34 mutants were decreased 2.9-65 fold, suggesting that the side chain of Ser34 supports catalysis by stabilizing the anionic oxygen of the proposed intermediate state (gem-diolate). This is the first crystal structure determination of CumD in an active form, with the Ser103 residue, one of the catalytically essential "triad", being intact. Improving the catalytic efficiency of a meta-cleavage product hydrolase (CumD) from Pseudomonas fluorescens IP01.,Jun SY, Fushinobu S, Nojiri H, Omori T, Shoun H, Wakagi T Biochim Biophys Acta. 2006 Jul;1764(7):1159-66. Epub 2006 Jun 7. PMID:16844437[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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