View source for 3hea


==The L29P/L124I mutation of Pseudomonas fluorescens esterase==
<StructureSection load='3hea' size='340' side='right'caption='[[3hea]], [[Resolution|resolution]] 1.90&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[3hea]] is a 6 chain structure with sequence from [https://en.wikipedia.org/wiki/Pseudomonas_fluorescens Pseudomonas fluorescens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3HEA OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=3HEA 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]] 1.9&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=EEE:ETHYL+ACETATE'>EEE</scene>, <scene name='pdbligand=GOL:GLYCEROL'>GOL</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=3hea FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3hea OCA], [https://pdbe.org/3hea PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=3hea RCSB], [https://www.ebi.ac.uk/pdbsum/3hea PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=3hea ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/ESTE_PSEFL ESTE_PSEFL] Bifunctional enzyme, capable of both ester hydrolysis and halogenation. Has a low bromoperoxidase activity. Acts on many phenolic esters.
== 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/he/3hea_consurf.spt"</scriptWhenChecked>
    <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview03.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=3hea ConSurf].
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== Publication Abstract from PubMed ==
Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation &gt;100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of epsilon-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k(cat), but K(m) also increased so the specificity constant, k(cat)/K(m), remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of epsilon-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for larger alcohol moieties but binds epsilon-caprolactone more tightly. These results are consistent with the natural function of perhydrolases being either hydrolysis of peroxycarboxylic acids or hydrolysis of lactones.

Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase (,).,Yin DL, Bernhardt P, Morley KL, Jiang Y, Cheeseman JD, Purpero V, Schrag JD, Kazlauskas RJ Biochemistry. 2010 Feb 10. PMID:20112920<ref>PMID:20112920</ref>

From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
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== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Large Structures]]
[[Category: Pseudomonas fluorescens]]
[[Category: Cheeseman JD]]
[[Category: Kazlauskas RJ]]
[[Category: Morley KL]]
[[Category: Schrag JD]]