6am7: Difference between revisions
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The | ==Engineered tryptophan synthase b-subunit from Pyrococcus furiosus, PfTrpB2B9== | ||
<StructureSection load='6am7' size='340' side='right' caption='[[6am7]], [[Resolution|resolution]] 1.47Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[6am7]] is a 4 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6AM7 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6AM7 FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=NA:SODIUM+ION'>NA</scene>, <scene name='pdbligand=PO4:PHOSPHATE+ION'>PO4</scene></td></tr> | |||
<tr id='NonStdRes'><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=LLP:(2S)-2-AMINO-6-[[3-HYDROXY-2-METHYL-5-(PHOSPHONOOXYMETHYL)PYRIDIN-4-YL]METHYLIDENEAMINO]HEXANOIC+ACID'>LLP</scene></td></tr> | |||
<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[5vm5|5vm5]]</td></tr> | |||
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Tryptophan_synthase Tryptophan synthase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.20 4.2.1.20] </span></td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6am7 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6am7 OCA], [http://pdbe.org/6am7 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6am7 RCSB], [http://www.ebi.ac.uk/pdbsum/6am7 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6am7 ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[[http://www.uniprot.org/uniprot/TRPB1_PYRFU TRPB1_PYRFU]] The beta subunit is responsible for the synthesis of L-tryptophan from indole and L-serine (By similarity). | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of non-canonical amino acids (ncAA). Previ-ously, we evolved the beta-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone cata-lyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the cata-lytic cycle. Concomitantly, the steady-state distribution of intermediates shifts to favor covalently bound Trp adducts, which is associated with increased thermodynamic stability of these species. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabi-lizes the large-scale motion of a sub-domain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to reca-pitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes. | |||
Directed evolution mimics allosteric activation by stepwise tuning of the conformational ensemble.,Buller AR, van Roye P, Cahn JKB, Scheele RA, Herger M, Arnold FH J Am Chem Soc. 2018 Apr 30. doi: 10.1021/jacs.8b03490. PMID:29712420<ref>PMID:29712420</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
<div class="pdbe-citations 6am7" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | |||
</StructureSection> | |||
[[Category: Tryptophan synthase]] | |||
[[Category: Buller, A R]] | |||
[[Category: Roye, P van]] | |||
[[Category: Allostery]] | |||
[[Category: Biosynthetic protein]] | |||
[[Category: Engineered]] | |||
[[Category: Plp type ii]] | |||
Revision as of 08:28, 16 May 2018
Engineered tryptophan synthase b-subunit from Pyrococcus furiosus, PfTrpB2B9Engineered tryptophan synthase b-subunit from Pyrococcus furiosus, PfTrpB2B9
Structural highlights
Function[TRPB1_PYRFU] The beta subunit is responsible for the synthesis of L-tryptophan from indole and L-serine (By similarity). Publication Abstract from PubMedAllosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of non-canonical amino acids (ncAA). Previ-ously, we evolved the beta-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone cata-lyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the cata-lytic cycle. Concomitantly, the steady-state distribution of intermediates shifts to favor covalently bound Trp adducts, which is associated with increased thermodynamic stability of these species. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabi-lizes the large-scale motion of a sub-domain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to reca-pitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes. Directed evolution mimics allosteric activation by stepwise tuning of the conformational ensemble.,Buller AR, van Roye P, Cahn JKB, Scheele RA, Herger M, Arnold FH J Am Chem Soc. 2018 Apr 30. doi: 10.1021/jacs.8b03490. PMID:29712420[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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