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==Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties== | |||
<StructureSection load='2ven' size='340' side='right' caption='[[2ven]], [[Resolution|resolution]] 2.00Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[2ven]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Trypanosoma_brucei_brucei Trypanosoma brucei brucei]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2VEN OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=2VEN FirstGlance]. <br> | |||
</td></tr><tr><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=CIT:CITRIC+ACID'>CIT</scene><br> | |||
<tr><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[1iih|1iih]], [[1kv5|1kv5]], [[1tpf|1tpf]], [[1trd|1trd]], [[1tsi|1tsi]], [[1tti|1tti]], [[1ttj|1ttj]], [[2j24|2j24]], [[2j27|2j27]], [[2v2c|2v2c]], [[2v2d|2v2d]], [[2v2h|2v2h]], [[2vei|2vei]], [[2vek|2vek]], [[1ag1|1ag1]], [[1dkw|1dkw]], [[1iig|1iig]], [[1ml1|1ml1]], [[1mss|1mss]], [[1mtm|1mtm]], [[1tpd|1tpd]], [[1tpe|1tpe]], [[1tri|1tri]], [[2v0t|2v0t]], [[2v5l|2v5l]], [[2vel|2vel]], [[4tim|4tim]], [[5tim|5tim]], [[6tim|6tim]], [[2vem|2vem]], [[3tim|3tim]]</td></tr> | |||
<tr><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Triose-phosphate_isomerase Triose-phosphate isomerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=5.3.1.1 5.3.1.1] </span></td></tr> | |||
<tr><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=2ven FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2ven OCA], [http://www.rcsb.org/pdb/explore.do?structureId=2ven RCSB], [http://www.ebi.ac.uk/pdbsum/2ven PDBsum]</span></td></tr> | |||
<table> | |||
== 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/ve/2ven_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/chain_selection.php?pdb_ID=2ata ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
A monomeric variant of triosephosphate isomerase (TIM) with a new engineered binding groove has been characterized further. In this variant (ml8bTIM), the phosphate binding loop had been shortened, causing the binding site to be much more extended. Here, it is reported that in the V233A variant of ml8bTIM (A-TIM), three important properties of the wild-type TIM active site have been restored: (i) the structural properties of loop-7, (ii) the binding site of a conserved water molecule between loop-7 and loop-8 and (iii) the binding site of the phosphate moiety. It is shown that the active site of A-TIM can bind TIM transition state analogs and suicide inhibitors competently. It is found that the active site geometry of the A-TIM complexes is less compact and more solvent exposed, as in wild-type TIM. This correlates with the observation that the catalytic efficiency of A-TIM for interconverting the TIM substrates is too low to be detected. It is also shown that the A-TIM active site can bind compounds which do not bind to wild-type TIM and which are completely different from the normal TIM substrate, like a citrate molecule. The binding of this citrate molecule is stabilized by hydrogen bonding interactions with the new binding groove. | |||
Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties.,Alahuhta M, Salin M, Casteleijn MG, Kemmer C, El-Sayed I, Augustyns K, Neubauer P, Wierenga RK Protein Eng Des Sel. 2008 Apr;21(4):257-66. Epub 2008 Jan 31. PMID:18239072<ref>PMID:18239072</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
==See Also== | ==See Also== | ||
*[[Triose Phosphate Isomerase|Triose Phosphate Isomerase]] | *[[Triose Phosphate Isomerase|Triose Phosphate Isomerase]] | ||
== References == | |||
== | <references/> | ||
< | __TOC__ | ||
</StructureSection> | |||
[[Category: Triose-phosphate isomerase]] | [[Category: Triose-phosphate isomerase]] | ||
[[Category: Trypanosoma brucei brucei]] | [[Category: Trypanosoma brucei brucei]] | ||
Revision as of 05:00, 1 October 2014
Structure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding propertiesStructure-based enzyme engineering efforts with an inactive monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties
Structural highlights
Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedA monomeric variant of triosephosphate isomerase (TIM) with a new engineered binding groove has been characterized further. In this variant (ml8bTIM), the phosphate binding loop had been shortened, causing the binding site to be much more extended. Here, it is reported that in the V233A variant of ml8bTIM (A-TIM), three important properties of the wild-type TIM active site have been restored: (i) the structural properties of loop-7, (ii) the binding site of a conserved water molecule between loop-7 and loop-8 and (iii) the binding site of the phosphate moiety. It is shown that the active site of A-TIM can bind TIM transition state analogs and suicide inhibitors competently. It is found that the active site geometry of the A-TIM complexes is less compact and more solvent exposed, as in wild-type TIM. This correlates with the observation that the catalytic efficiency of A-TIM for interconverting the TIM substrates is too low to be detected. It is also shown that the A-TIM active site can bind compounds which do not bind to wild-type TIM and which are completely different from the normal TIM substrate, like a citrate molecule. The binding of this citrate molecule is stabilized by hydrogen bonding interactions with the new binding groove. Structure-based protein engineering efforts with a monomeric TIM variant: the importance of a single point mutation for generating an active site with suitable binding properties.,Alahuhta M, Salin M, Casteleijn MG, Kemmer C, El-Sayed I, Augustyns K, Neubauer P, Wierenga RK Protein Eng Des Sel. 2008 Apr;21(4):257-66. Epub 2008 Jan 31. PMID:18239072[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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