1ocv: Difference between revisions
No edit summary |
No edit summary |
||
| (8 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
==the F116W mutant structure of ketosteroid isomerase from Comamonas testosteroni== | |||
<StructureSection load='1ocv' size='340' side='right'caption='[[1ocv]], [[Resolution|resolution]] 2.00Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[1ocv]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Comamonas_testosteroni Comamonas testosteroni]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1OCV OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1OCV 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]] 2Å</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=1ocv FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1ocv OCA], [https://pdbe.org/1ocv PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1ocv RCSB], [https://www.ebi.ac.uk/pdbsum/1ocv PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1ocv ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/SDIS_COMTE SDIS_COMTE] | |||
== 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/oc/1ocv_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=1ocv ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKa value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering. | |||
Origin of the different pH activity profile in two homologous ketosteroid isomerases.,Yun YS, Lee TH, Nam GH, Jang DS, Shin S, Oh BH, Choi KY J Biol Chem. 2003 Jul 25;278(30):28229-36. Epub 2003 May 6. PMID:12734184<ref>PMID:12734184</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 1ocv" style="background-color:#fffaf0;"></div> | |||
== | ==See Also== | ||
[[ | *[[Ketosteroid Isomerase|Ketosteroid Isomerase]] | ||
== References == | |||
== | <references/> | ||
< | __TOC__ | ||
</StructureSection> | |||
[[Category: Comamonas testosteroni]] | [[Category: Comamonas testosteroni]] | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: Lee | [[Category: Lee T-H]] | ||
[[Category: Shin | [[Category: Shin S]] | ||
[[Category: Yun | [[Category: Yun YS]] | ||
Latest revision as of 15:35, 13 December 2023
the F116W mutant structure of ketosteroid isomerase from Comamonas testosteronithe F116W mutant structure of ketosteroid isomerase from Comamonas testosteroni
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 PubMedTwo homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKa value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering. Origin of the different pH activity profile in two homologous ketosteroid isomerases.,Yun YS, Lee TH, Nam GH, Jang DS, Shin S, Oh BH, Choi KY J Biol Chem. 2003 Jul 25;278(30):28229-36. Epub 2003 May 6. PMID:12734184[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
|
| ||||||||||||||||