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[[Image:1ea2.gif|left|200px]]
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{{STRUCTURE_1ea2|  PDB=1ea2  |  SCENE=  }}
'''PSEUDOREVERSION OF THE CATALYTIC ACTIVITY OF Y14F BY THE ADDITIONAL TYROSIN-TO-PHENYLALANINE SUBSTITUTION(S) IN THE HYDROGEN BOND NETWORK OF DELTA-5-3-KETOSTEROID ISOMERASE FROM PHEUDOMONAS PUTIDA BIOTYPE B'''


==Pseudoreversion of the Catalytic Activity of Y14F by the Additional Tyrosine-to-Phenylalanine Substitution(s) in the Hydrogen Bond Network of Delta-5-3-Ketosteroid Isomerase from Pheudomonas putida Biotype B==
<StructureSection load='1ea2' size='340' side='right'caption='[[1ea2]], [[Resolution|resolution]] 1.80&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[1ea2]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Pseudomonas_putida Pseudomonas putida]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1EA2 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1EA2 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]] 1.8&#8491;</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=1ea2 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1ea2 OCA], [https://pdbe.org/1ea2 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1ea2 RCSB], [https://www.ebi.ac.uk/pdbsum/1ea2 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1ea2 ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/SDIS_PSEPU SDIS_PSEPU]
== 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/ea/1ea2_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=1ea2 ConSurf].
<div style="clear:both"></div>
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Delta5-3-ketosteroid isomerase (KSI) from Pseudomonas putida Biotype B catalyzes the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers via a dienolate intermediate. Two electrophilic catalysts, Tyr-14 and Asp-99, are involved in a hydrogen bond network that comprises Asp-99 Odelta2...O of Wat504...Tyr-14 Oeta...Tyr-55 Oeta.Tyr-30 Oeta in the active site of P. putida KSI. Even though neither Tyr-30 nor Tyr-55 plays an essential role in catalysis by the KSI, the catalytic activity of Y14F could be increased ca. 26-51-fold by the additional Y30F and/or Y55F mutation in the hydrogen bond network. To identify the structural basis for the pseudoreversion in the KSI, crystal structures of Y14F and Y14F/Y30F/Y55F have been determined at 1.8 and 2.0 A resolution, respectively. Comparisons of the two structures near the catalytic center indicate that the hydrogen bond between Asp-99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by the additional Y30F/Y55F mutations. The kinetic parameters of the tyrosine mutants with the additional D99N or D99L mutation also support the idea that Asp-99 contributes to catalysis more efficiently in Y14F/Y30F/Y55F than in Y14F. In contrast to the catalytic mechanism of Y14F, the C4 proton of the steroid substrate was found to be transferred to the C6 position in Y14F/Y30F/Y55F with little exchange of the substrate 4beta-proton with a solvent deuterium based on the reaction rate in D2O. Taken together, our findings strongly suggest that the improvement in the catalytic activity of Y14F by the additional Y30F/Y55F mutations is due to the changes in the structural integrity at the catalytic site and the resulting restoration of the proton-transfer mechanism in Y14F/Y30F/Y55F.


==Overview==
Pseudoreversion of the catalytic activity of Y14F by the additional substitution(s) of tyrosine with phenylalanine in the hydrogen bond network of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.,Choi G, Ha NC, Kim MS, Hong BH, Oh BH, Choi KY Biochemistry. 2001 Jun 12;40(23):6828-35. PMID:11389596<ref>PMID:11389596</ref>
Delta5-3-ketosteroid isomerase (KSI) from Pseudomonas putida Biotype B catalyzes the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers via a dienolate intermediate. Two electrophilic catalysts, Tyr-14 and Asp-99, are involved in a hydrogen bond network that comprises Asp-99 Odelta2...O of Wat504...Tyr-14 Oeta...Tyr-55 Oeta.Tyr-30 Oeta in the active site of P. putida KSI. Even though neither Tyr-30 nor Tyr-55 plays an essential role in catalysis by the KSI, the catalytic activity of Y14F could be increased ca. 26-51-fold by the additional Y30F and/or Y55F mutation in the hydrogen bond network. To identify the structural basis for the pseudoreversion in the KSI, crystal structures of Y14F and Y14F/Y30F/Y55F have been determined at 1.8 and 2.0 A resolution, respectively. Comparisons of the two structures near the catalytic center indicate that the hydrogen bond between Asp-99 Odelta2 and C3-O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type enzyme by the additional Y30F/Y55F mutations. The kinetic parameters of the tyrosine mutants with the additional D99N or D99L mutation also support the idea that Asp-99 contributes to catalysis more efficiently in Y14F/Y30F/Y55F than in Y14F. In contrast to the catalytic mechanism of Y14F, the C4 proton of the steroid substrate was found to be transferred to the C6 position in Y14F/Y30F/Y55F with little exchange of the substrate 4beta-proton with a solvent deuterium based on the reaction rate in D2O. Taken together, our findings strongly suggest that the improvement in the catalytic activity of Y14F by the additional Y30F/Y55F mutations is due to the changes in the structural integrity at the catalytic site and the resulting restoration of the proton-transfer mechanism in Y14F/Y30F/Y55F.


==About this Structure==
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
1EA2 is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Pseudomonas_putida Pseudomonas putida]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1EA2 OCA].
</div>
<div class="pdbe-citations 1ea2" style="background-color:#fffaf0;"></div>


==Reference==
==See Also==
Pseudoreversion of the catalytic activity of Y14F by the additional substitution(s) of tyrosine with phenylalanine in the hydrogen bond network of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B., Choi G, Ha NC, Kim MS, Hong BH, Oh BH, Choi KY, Biochemistry. 2001 Jun 12;40(23):6828-35. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/11389596 11389596]
*[[Ketosteroid Isomerase|Ketosteroid Isomerase]]
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Large Structures]]
[[Category: Pseudomonas putida]]
[[Category: Pseudomonas putida]]
[[Category: Single protein]]
[[Category: Choi G]]
[[Category: Choi, G.]]
[[Category: Choi KY]]
[[Category: Choi, K.]]
[[Category: Ha N-C]]
[[Category: Ha, N C.]]
[[Category: Hong B-H]]
[[Category: Hong, B H.]]
[[Category: Kim M-S]]
[[Category: Kim, M S.]]
[[Category: Oh, B H.]]
[[Category: Ketosteroid isomerase]]
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May  2 14:51:20 2008''
Retrieved from "https://proteopedia.org/1ea2"