1b97: Difference between revisions

New page: left|200px<br /><applet load="1b97" size="450" color="white" frame="true" align="right" spinBox="true" caption="1b97, resolution 1.90Å" /> '''ANALYSIS OF A MUTATI...
 
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[[Image:1b97.gif|left|200px]]<br /><applet load="1b97" size="450" color="white" frame="true" align="right" spinBox="true"
caption="1b97, resolution 1.90&Aring;" />
'''ANALYSIS OF A MUTATIONAL HOT-SPOT IN THE ECORV RESTRICTION ENDONUCLEASE: A CATALYTIC ROLE FOR A MAIN CHAIN CARBONYL GROUP'''<br />


==Overview==
==ANALYSIS OF A MUTATIONAL HOT-SPOT IN THE ECORV RESTRICTION ENDONUCLEASE: A CATALYTIC ROLE FOR A MAIN CHAIN CARBONYL GROUP==
Following random mutagenesis of the Eco RV endonuclease, a high proportion, of the null mutants carry substitutions at Gln69. Such mutants display, reduced rates for the DNA cleavage step in the reaction pathway, yet the, crystal structures of wild-type Eco RV fail to explain why Gln69 is, crucial for activity. In this study, crystal structures were determined, for two mutants of Eco RV, with Leu or Glu at residue 69, bound to, specific DNA. The structures of the mutants are similar to the native, protein and no function can be ascribed to the side chain of the amino, acid at this locus. Instead, the structures of the mutant proteins suggest, that the catalytic defect is due to the positioning of the main chain, carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain, carbonyl of Gln69 makes no interactions with catalytic functions but, in, the enzyme-product complex, it coordinates a metal ion bound to the newly, liberated 5'-phosphate. This re-positioning may be hindered in the mutant, proteins. Molecular dynamics calculations indicate that the metal on the, phosphoryl oxygen interacts with the carbonyl group upon forming the, pentavalent intermediate during phosphodiester hydrolysis. A main chain, carbonyl may thus play a role in catalysis by Eco RV.
<StructureSection load='1b97' size='340' side='right'caption='[[1b97]], [[Resolution|resolution]] 1.90&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[1b97]] is a 4 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=1B97 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1B97 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='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1b97 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1b97 OCA], [https://pdbe.org/1b97 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1b97 RCSB], [https://www.ebi.ac.uk/pdbsum/1b97 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1b97 ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/T2E5_ECOLX T2E5_ECOLX] Recognizes the double-stranded sequence GATATC and cleaves after T-3.
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.


==About this Structure==
Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.,Thomas MP, Brady RL, Halford SE, Sessions RB, Baldwin GS Nucleic Acids Res. 1999 Sep 1;27(17):3438-45. PMID:10446231<ref>PMID:10446231</ref>
1B97 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Active as [http://en.wikipedia.org/wiki/Type_II_site-specific_deoxyribonuclease Type II site-specific deoxyribonuclease], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.21.4 3.1.21.4] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1B97 OCA].


==Reference==
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group., Thomas MP, Brady RL, Halford SE, Sessions RB, Baldwin GS, Nucleic Acids Res. 1999 Sep 1;27(17):3438-45. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=10446231 10446231]
</div>
<div class="pdbe-citations 1b97" style="background-color:#fffaf0;"></div>
 
==See Also==
*[[Endonuclease 3D structures|Endonuclease 3D structures]]
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Escherichia coli]]
[[Category: Escherichia coli]]
[[Category: Single protein]]
[[Category: Large Structures]]
[[Category: Type II site-specific deoxyribonuclease]]
[[Category: Brady RL]]
[[Category: Brady, R.L.]]
[[Category: Halford SE]]
[[Category: Halford, S.E.]]
[[Category: Thomas MP]]
[[Category: Thomas, M.P.]]
[[Category: ecorv]]
[[Category: endonuclease]]
[[Category: restriction]]
 
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