1toe: Difference between revisions
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< | ==Unliganded structure of Hexamutant + A293D mutant of E. coli aspartate aminotransferase== | ||
<StructureSection load='1toe' size='340' side='right'caption='[[1toe]], [[Resolution|resolution]] 2.00Å' scene=''> | |||
You may | == Structural highlights == | ||
or the | <table><tr><td colspan='2'>[[1toe]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1TOE OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1TOE 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='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=LLP:(2S)-2-AMINO-6-[[3-HYDROXY-2-METHYL-5-(PHOSPHONOOXYMETHYL)PYRIDIN-4-YL]METHYLIDENEAMINO]HEXANOIC+ACID'>LLP</scene>, <scene name='pdbligand=SO4:SULFATE+ION'>SO4</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=1toe FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1toe OCA], [https://pdbe.org/1toe PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1toe RCSB], [https://www.ebi.ac.uk/pdbsum/1toe PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1toe ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[https://www.uniprot.org/uniprot/AAT_ECOLI AAT_ECOLI] | |||
== 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/to/1toe_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=1toe ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Several mutant Escherichia coli aspartate aminotransferases (eAATases) have been characterized in the attempt to evolve or rationally redesign the substrate specificity of eAATase into that of E. coli tyrosine aminotransferase (eTATase). These include HEX (designed), HEX + A293D (design followed by directed evolution), and SRHEPT (directed evolution). The A293D mutation realized from directed evolution of HEX is here imported into the SRHEPT platform by site-directed mutagenesis, resulting in an enzyme (SRHEPT + A293D) with nearly the same ratio of k(cat)/K(m)(Phe) to k(cat)/K(m)(Asp) as that of wild-type eTATase. The A293D substitution is an important specificity determinant; it selectively disfavors interactions with dicarboxylic substrates and inhibitors compared to aromatic ones. Context dependence analysis is generalized to provide quantitative comparisons of a common substitution in two or more different protein scaffolds. High-resolution crystal structures of ligand complexes of HEX + A293D, SRHEPT, and SRHEPT + A293D were determined. We find that in both SRHEPT + A293D and HEX + A293D, the additional mutation holds the Arg 292 side chain away from the active site to allow increased specificity for phenylalanine over aspartate. The resulting movement of Arg 292 allows greater flexibility of the small domain in HEX + A293D. While HEX is always in the closed conformation, HEX + A293D is observed in both the closed and a novel open conformation, allowing for more rapid product release. | |||
Narrowing substrate specificity in a directly evolved enzyme: the A293D mutant of aspartate aminotransferase.,Chow MA, McElroy KE, Corbett KD, Berger JM, Kirsch JF Biochemistry. 2004 Oct 12;43(40):12780-7. PMID:15461450<ref>PMID:15461450</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 1toe" style="background-color:#fffaf0;"></div> | |||
==See Also== | |||
*[[Aspartate aminotransferase 3D structures|Aspartate aminotransferase 3D structures]] | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
== | |||
== | |||
[[Category: Escherichia coli]] | [[Category: Escherichia coli]] | ||
[[Category: | [[Category: Large Structures]] | ||
[[Category: Berger | [[Category: Berger JM]] | ||
[[Category: Chow | [[Category: Chow MA]] | ||
[[Category: Corbett | [[Category: Corbett KD]] | ||
[[Category: Kirsch | [[Category: Kirsch JF]] | ||
[[Category: McElroy | [[Category: McElroy KE]] | ||
Latest revision as of 09:32, 23 August 2023
Unliganded structure of Hexamutant + A293D mutant of E. coli aspartate aminotransferaseUnliganded structure of Hexamutant + A293D mutant of E. coli aspartate aminotransferase
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 PubMedSeveral mutant Escherichia coli aspartate aminotransferases (eAATases) have been characterized in the attempt to evolve or rationally redesign the substrate specificity of eAATase into that of E. coli tyrosine aminotransferase (eTATase). These include HEX (designed), HEX + A293D (design followed by directed evolution), and SRHEPT (directed evolution). The A293D mutation realized from directed evolution of HEX is here imported into the SRHEPT platform by site-directed mutagenesis, resulting in an enzyme (SRHEPT + A293D) with nearly the same ratio of k(cat)/K(m)(Phe) to k(cat)/K(m)(Asp) as that of wild-type eTATase. The A293D substitution is an important specificity determinant; it selectively disfavors interactions with dicarboxylic substrates and inhibitors compared to aromatic ones. Context dependence analysis is generalized to provide quantitative comparisons of a common substitution in two or more different protein scaffolds. High-resolution crystal structures of ligand complexes of HEX + A293D, SRHEPT, and SRHEPT + A293D were determined. We find that in both SRHEPT + A293D and HEX + A293D, the additional mutation holds the Arg 292 side chain away from the active site to allow increased specificity for phenylalanine over aspartate. The resulting movement of Arg 292 allows greater flexibility of the small domain in HEX + A293D. While HEX is always in the closed conformation, HEX + A293D is observed in both the closed and a novel open conformation, allowing for more rapid product release. Narrowing substrate specificity in a directly evolved enzyme: the A293D mutant of aspartate aminotransferase.,Chow MA, McElroy KE, Corbett KD, Berger JM, Kirsch JF Biochemistry. 2004 Oct 12;43(40):12780-7. PMID:15461450[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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