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| | <StructureSection load='6t8e' size='340' side='right'caption='[[6t8e]], [[Resolution|resolution]] 1.86Å' scene=''> | | <StructureSection load='6t8e' size='340' side='right'caption='[[6t8e]], [[Resolution|resolution]] 1.86Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[6t8e]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Pirse Pirse]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6T8E OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6T8E FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[6t8e]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Piromyces_sp._E2 Piromyces sp. E2]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6T8E OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6T8E FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene>, <scene name='pdbligand=XLS:D-XYLOSE+(LINEAR+FORM)'>XLS</scene>, <scene name='pdbligand=XYP:BETA-D-XYLOPYRANOSE'>XYP</scene>, <scene name='pdbligand=XYS:XYLOPYRANOSE'>XYS</scene></td></tr> | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.86Å</td></tr> |
| - | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">xylA ([https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=73868 PIRSE])</td></tr>
| + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</scene>, <scene name='pdbligand=XLS:D-XYLOSE+(LINEAR+FORM)'>XLS</scene>, <scene name='pdbligand=XYP:BETA-D-XYLOPYRANOSE'>XYP</scene>, <scene name='pdbligand=XYS:XYLOPYRANOSE'>XYS</scene></td></tr> |
| - | <tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[https://en.wikipedia.org/wiki/Xylose_isomerase Xylose isomerase], with EC number [https://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.3.1.5 5.3.1.5] </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=6t8e FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6t8e OCA], [https://pdbe.org/6t8e PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6t8e RCSB], [https://www.ebi.ac.uk/pdbsum/6t8e PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6t8e ProSAT]</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=6t8e FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6t8e OCA], [https://pdbe.org/6t8e PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6t8e RCSB], [https://www.ebi.ac.uk/pdbsum/6t8e PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6t8e ProSAT]</span></td></tr> |
| | </table> | | </table> |
| | + | == Function == |
| | + | [https://www.uniprot.org/uniprot/Q9P8C9_PIRSE Q9P8C9_PIRSE] |
| | <div style="background-color:#fffaf0;"> | | <div style="background-color:#fffaf0;"> |
| | == Publication Abstract from PubMed == | | == Publication Abstract from PubMed == |
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| | </StructureSection> | | </StructureSection> |
| | [[Category: Large Structures]] | | [[Category: Large Structures]] |
| - | [[Category: Pirse]] | + | [[Category: Piromyces sp. E2]] |
| - | [[Category: Xylose isomerase]]
| + | [[Category: Janssen DB]] |
| - | [[Category: Janssen, D B]] | + | [[Category: Rozeboom HJ]] |
| - | [[Category: Rozeboom, H J]] | + | |
| - | [[Category: Isomerase]]
| + | |
| - | [[Category: Tim-barrel]]
| + | |
| Structural highlights
6t8e is a 4 chain structure with sequence from Piromyces sp. E2. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
| | Method: | X-ray diffraction, Resolution 1.86Å |
| Ligands: | , , , , |
| Resources: | FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT |
Function
Q9P8C9_PIRSE
Publication Abstract from PubMed
Background: Efficient bioethanol production from hemicellulose feedstocks by Saccharomyces cerevisiae requires xylose utilization. Whereas S. cerevisiae does not metabolize xylose, engineered strains that express xylose isomerase can metabolize xylose by converting it to xylulose. For this, the type II xylose isomerase from Piromyces (PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts. Results: We constructed focused mutant libraries of Piromyces xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to S. cerevisiae and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal. Conclusion: This study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism.
Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance.,Lee M, Rozeboom HJ, Keuning E, de Waal P, Janssen DB Biotechnol Biofuels. 2020 Jan 11;13:5. doi: 10.1186/s13068-019-1643-0., eCollection 2020. PMID:31938040[1]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Lee M, Rozeboom HJ, Keuning E, de Waal P, Janssen DB. Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance. Biotechnol Biofuels. 2020 Jan 11;13:5. doi: 10.1186/s13068-019-1643-0., eCollection 2020. PMID:31938040 doi:http://dx.doi.org/10.1186/s13068-019-1643-0
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