3gtf: Difference between revisions
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< | ==D71G/E101G/V235L mutant in organophosphorus hydrolase from Deinococcus radiodurans== | ||
<StructureSection load='3gtf' size='340' side='right'caption='[[3gtf]], [[Resolution|resolution]] 1.98Å' scene=''> | |||
You may | == Structural highlights == | ||
<table><tr><td colspan='2'>[[3gtf]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/"micrococcus_radiodurans"_raj_et_al._1960 "micrococcus radiodurans" raj et al. 1960]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3GTF OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3GTF FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CO:COBALT+(II)+ION'>CO</scene></td></tr> | |||
-- | <tr id='NonStdRes'><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=KCX:LYSINE+NZ-CARBOXYLIC+ACID'>KCX</scene></td></tr> | ||
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">DR_0930 ([https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=1299 "Micrococcus radiodurans" Raj et al. 1960])</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=3gtf FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3gtf OCA], [https://pdbe.org/3gtf PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3gtf RCSB], [https://www.ebi.ac.uk/pdbsum/3gtf PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3gtf ProSAT]</span></td></tr> | |||
</table> | |||
== 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/gt/3gtf_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=3gtf ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 degrees C and withstanding incubation at temperatures up to 70 degrees C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 A. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those factors most important for maintaining the structure and function of the enzyme. | |||
Structure-based and random mutagenesis approaches increase the organophosphate-degrading activity of a phosphotriesterase homologue from Deinococcus radiodurans.,Hawwa R, Larsen SD, Ratia K, Mesecar AD J Mol Biol. 2009 Oct 16;393(1):36-57. Epub 2009 Jul 22. PMID:19631223<ref>PMID:19631223</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 3gtf" style="background-color:#fffaf0;"></div> | |||
== | ==See Also== | ||
*[[Phosphotriesterase 3D structures|Phosphotriesterase 3D structures]] | |||
[[Category: | == References == | ||
[[Category: Hawwa, R | <references/> | ||
[[Category: Larsen, S | __TOC__ | ||
[[Category: Mesecar, A | </StructureSection> | ||
[[Category: Ratia, K | [[Category: Micrococcus radiodurans raj et al. 1960]] | ||
[[Category: Large Structures]] | |||
[[Category: Hawwa, R]] | |||
[[Category: Larsen, S]] | |||
[[Category: Mesecar, A]] | |||
[[Category: Ratia, K]] | |||
[[Category: Alpha-beta barrel]] | [[Category: Alpha-beta barrel]] | ||
[[Category: Amidohydrolase]] | [[Category: Amidohydrolase]] | ||
[[Category: Hydrolase]] | |||
[[Category: Mutant]] | [[Category: Mutant]] | ||
Latest revision as of 23:10, 20 October 2021
D71G/E101G/V235L mutant in organophosphorus hydrolase from Deinococcus radioduransD71G/E101G/V235L mutant in organophosphorus hydrolase from Deinococcus radiodurans
Structural highlights
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 PubMedAn enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 degrees C and withstanding incubation at temperatures up to 70 degrees C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 A. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those factors most important for maintaining the structure and function of the enzyme. Structure-based and random mutagenesis approaches increase the organophosphate-degrading activity of a phosphotriesterase homologue from Deinococcus radiodurans.,Hawwa R, Larsen SD, Ratia K, Mesecar AD J Mol Biol. 2009 Oct 16;393(1):36-57. Epub 2009 Jul 22. PMID:19631223[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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