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==Closed form of PET-degrading cutinase Cut190 with thermostability-improving mutations of S226P/R228S/Q138A/D250C-E296C/Q123H/N202H== | ==Closed form of PET-degrading cutinase Cut190 with thermostability-improving mutations of S226P/R228S/Q138A/D250C-E296C/Q123H/N202H== | ||
<StructureSection load='7ctr' size='340' side='right'caption='[[7ctr]]' scene=''> | <StructureSection load='7ctr' size='340' side='right'caption='[[7ctr]], [[Resolution|resolution]] 1.20Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=7CTR OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7CTR FirstGlance]. <br> | <table><tr><td colspan='2'>[[7ctr]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/"saccharomonospora_internatus"_(agre_et_al._1974)_greiner-mai_et_al._1988 "saccharomonospora internatus" (agre et al. 1974) greiner-mai et al. 1988]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=7CTR OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7CTR FirstGlance]. <br> | ||
</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=7ctr FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7ctr OCA], [https://pdbe.org/7ctr PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7ctr RCSB], [https://www.ebi.ac.uk/pdbsum/7ctr PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7ctr ProSAT]</span></td></tr> | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=DIO:1,4-DIETHYLENE+DIOXIDE'>DIO</scene></td></tr> | ||
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">Cut190, SAMN02982918_2340 ([https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=1852 "Saccharomonospora internatus" (Agre et al. 1974) Greiner-Mai et al. 1988])</td></tr> | |||
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[https://en.wikipedia.org/wiki/Cutinase Cutinase], with EC number [https://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.1.74 3.1.1.74] </span></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=7ctr FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7ctr OCA], [https://pdbe.org/7ctr PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7ctr RCSB], [https://www.ebi.ac.uk/pdbsum/7ctr PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7ctr ProSAT]</span></td></tr> | |||
</table> | </table> | ||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
The cutinase-like enzyme from the thermophile Saccharomonospora viridis AHK190, Cut190, is a good candidate to depolymerize polyethylene terephthalate (PET) efficiently. We previously developed a mutant of Cut190 (S226P/R228S), which we designated as Cut190* that has both increased activity and stability and solved its crystal structure. Recently, we showed that mutation of D250C/E296C on one of the Ca(2+) -binding sites resulted in a higher thermal stability while retaining its polyesterase activity. In this study, we solved the crystal structures of Cut190* mutants, Q138A/D250C-E296C/Q123H/N202H, designated as Cut190*SS, and its inactive S176A mutant, Cut190*SS_S176A, at high resolution. The overall structures were similar to those of Cut190* and Cut190*S176A reported previously. As expected, Cys250 and Cys296 were closely located to form a disulfide bond, which would assuredly contribute to increase the stability. Isothermal titration calorimetry experiments and 3D Reference Interaction Site Model calculations showed that the metal-binding properties of the Cut190*SS series were different from those of the Cut190* series. However, our results show that binding of Ca(2+) to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca(2+) to accelerate the conformational change from the closed (inactive) to the open (active) form. While increasing the thermal stability, Cut190*SS could still express its enzymatic function. Even after incubation at 70 degrees C, which corresponds to the glass transition temperature of PET, the enzyme retained its activity well, implying a high applicability for industrial PET depolymerization using Cut190*SS. | |||
Structural basis of mutants of PET-degrading enzyme from Saccharomonospora viridis AHK190 with high activity and thermal stability.,Emori M, Numoto N, Senga A, Bekker GJ, Kamiya N, Kobayashi Y, Ito N, Kawai F, Oda M Proteins. 2020 Dec 19. doi: 10.1002/prot.26034. PMID:33340163<ref>PMID:33340163</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 7ctr" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | __TOC__ | ||
</StructureSection> | </StructureSection> | ||
[[Category: Cutinase]] | |||
[[Category: Large Structures]] | [[Category: Large Structures]] | ||
[[Category: Bekker | [[Category: Bekker, G J]] | ||
[[Category: Emori M]] | [[Category: Emori, M]] | ||
[[Category: Ito N]] | [[Category: Ito, N]] | ||
[[Category: Kamiya N]] | [[Category: Kamiya, N]] | ||
[[Category: Kawai F]] | [[Category: Kawai, F]] | ||
[[Category: Numoto N]] | [[Category: Numoto, N]] | ||
[[Category: Oda M]] | [[Category: Oda, M]] | ||
[[Category: Senga A]] | [[Category: Senga, A]] | ||
[[Category: Disulfide bond]] | |||
[[Category: Hydrolase]] | |||
[[Category: Metal binding]] | |||
[[Category: Polyesterase]] | |||
[[Category: Protein engineering]] | |||
Revision as of 10:02, 14 April 2021
Closed form of PET-degrading cutinase Cut190 with thermostability-improving mutations of S226P/R228S/Q138A/D250C-E296C/Q123H/N202HClosed form of PET-degrading cutinase Cut190 with thermostability-improving mutations of S226P/R228S/Q138A/D250C-E296C/Q123H/N202H
Structural highlights
Publication Abstract from PubMedThe cutinase-like enzyme from the thermophile Saccharomonospora viridis AHK190, Cut190, is a good candidate to depolymerize polyethylene terephthalate (PET) efficiently. We previously developed a mutant of Cut190 (S226P/R228S), which we designated as Cut190* that has both increased activity and stability and solved its crystal structure. Recently, we showed that mutation of D250C/E296C on one of the Ca(2+) -binding sites resulted in a higher thermal stability while retaining its polyesterase activity. In this study, we solved the crystal structures of Cut190* mutants, Q138A/D250C-E296C/Q123H/N202H, designated as Cut190*SS, and its inactive S176A mutant, Cut190*SS_S176A, at high resolution. The overall structures were similar to those of Cut190* and Cut190*S176A reported previously. As expected, Cys250 and Cys296 were closely located to form a disulfide bond, which would assuredly contribute to increase the stability. Isothermal titration calorimetry experiments and 3D Reference Interaction Site Model calculations showed that the metal-binding properties of the Cut190*SS series were different from those of the Cut190* series. However, our results show that binding of Ca(2+) to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca(2+) to accelerate the conformational change from the closed (inactive) to the open (active) form. While increasing the thermal stability, Cut190*SS could still express its enzymatic function. Even after incubation at 70 degrees C, which corresponds to the glass transition temperature of PET, the enzyme retained its activity well, implying a high applicability for industrial PET depolymerization using Cut190*SS. Structural basis of mutants of PET-degrading enzyme from Saccharomonospora viridis AHK190 with high activity and thermal stability.,Emori M, Numoto N, Senga A, Bekker GJ, Kamiya N, Kobayashi Y, Ito N, Kawai F, Oda M Proteins. 2020 Dec 19. doi: 10.1002/prot.26034. PMID:33340163[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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