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==Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI==
==Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI==
<StructureSection load='2i9k' size='340' side='right' caption='[[2i9k]], [[Resolution|resolution]] 2.65&Aring;' scene=''>
<StructureSection load='2i9k' size='340' side='right'caption='[[2i9k]], [[Resolution|resolution]] 2.65&Aring;' scene=''>
== Structural highlights ==
== Structural highlights ==
<table><tr><td colspan='2'>[[2i9k]] is a 3 chain structure with sequence from [http://en.wikipedia.org/wiki/"bacillus_x"_pritchett_and_stillman_1919 "bacillus x" pritchett and stillman 1919]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2I9K OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=2I9K FirstGlance]. <br>
<table><tr><td colspan='2'>[[2i9k]] is a 3 chain structure with sequence from [https://en.wikipedia.org/wiki/Haemophilus_haemolyticus Haemophilus haemolyticus]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2I9K OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2I9K FirstGlance]. <br>
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=SAH:S-ADENOSYL-L-HOMOCYSTEINE'>SAH</scene></td></tr>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.65&#8491;</td></tr>
<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[2hr1|2hr1]], [[3mht|3mht]]</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=SAH:S-ADENOSYL-L-HOMOCYSTEINE'>SAH</scene></td></tr>
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">hhaIM ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=726 "Bacillus X" Pritchett and Stillman 1919])</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=2i9k FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2i9k OCA], [https://pdbe.org/2i9k PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2i9k RCSB], [https://www.ebi.ac.uk/pdbsum/2i9k PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2i9k ProSAT]</span></td></tr>
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/DNA_(cytosine-5-)-methyltransferase DNA (cytosine-5-)-methyltransferase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.1.1.37 2.1.1.37] </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=2i9k FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2i9k OCA], [http://pdbe.org/2i9k PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=2i9k RCSB], [http://www.ebi.ac.uk/pdbsum/2i9k PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=2i9k ProSAT]</span></td></tr>
</table>
</table>
== Function ==
== Function ==
[[http://www.uniprot.org/uniprot/MTH1_HAEPH MTH1_HAEPH]] This methylase recognizes the double-stranded sequence GCGC, causes specific methylation on C-2 on both strands, and protects the DNA from cleavage by the HhaI endonuclease.  
[https://www.uniprot.org/uniprot/MTH1_HAEPH MTH1_HAEPH] This methylase recognizes the double-stranded sequence GCGC, causes specific methylation on C-2 on both strands, and protects the DNA from cleavage by the HhaI endonuclease.
== Evolutionary Conservation ==
== Evolutionary Conservation ==
[[Image:Consurf_key_small.gif|200px|right]]
[[Image:Consurf_key_small.gif|200px|right]]
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</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=2i9k ConSurf].
</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=2i9k ConSurf].
<div style="clear:both"></div>
<div style="clear:both"></div>
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Improved sequence specificity of the DNA cytosine methyltransferase HhaI was achieved by disrupting interactions at a hydrophobic interface between the active site of the enzyme and a highly conserved flexible loop. Transient fluorescence experiments show that mutations disrupting this interface destabilize the positioning of the extrahelical, "flipped" cytosine base within the active site. The ternary crystal structure of the F124A M.HhaI bound to cognate DNA and the cofactor analogue S-adenosyl-l-homocysteine shows an increase in cavity volume between the flexible loop and the core of the enzyme. This cavity disrupts the interface between the loop and the active site, thereby destabilizing the extrahelical target base. The favored partitioning of the base-flipped enzyme-DNA complex back to the base-stacked intermediate results in the mutant enzyme discriminating better than the wild-type enzyme against non-cognate sites. Building upon the concepts of kinetic proofreading and our understanding of M.HhaI, we describe how a 16-fold specificity enhancement achieved with a double mutation at the loop/active site interface is acquired through destabilization of intermediates prior to methyltransfer rather than disruption of direct interactions between the enzyme and the substrate for M.HhaI.
Engineered extrahelical base destabilization enhances sequence discrimination of DNA methyltransferase M.HhaI.,Youngblood B, Shieh FK, De Los Rios S, Perona JJ, Reich NO J Mol Biol. 2006 Sep 15;362(2):334-46. Epub 2006 Jul 21. PMID:16919299<ref>PMID:16919299</ref>
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
</div>
<div class="pdbe-citations 2i9k" style="background-color:#fffaf0;"></div>


==See Also==
==See Also==
*[[DNA methyltransferase|DNA methyltransferase]]
*[[DNA methyltransferase 3D structures|DNA methyltransferase 3D structures]]
== References ==
<references/>
__TOC__
__TOC__
</StructureSection>
</StructureSection>
[[Category: Bacillus x pritchett and stillman 1919]]
[[Category: Haemophilus haemolyticus]]
[[Category: Perona, J J]]
[[Category: Large Structures]]
[[Category: Reich, N O]]
[[Category: De Los Rios S]]
[[Category: Rios, S De Los]]
[[Category: Perona JJ]]
[[Category: Shieh, F K]]
[[Category: Reich NO]]
[[Category: Youngblood, B]]
[[Category: Shieh FK]]
[[Category: Phe124ala mutation in m hhai]]
[[Category: Youngblood B]]
[[Category: Transferase-dna complex]]

Latest revision as of 12:02, 21 February 2024

Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaIEngineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI

Structural highlights

2i9k is a 3 chain structure with sequence from Haemophilus haemolyticus. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:X-ray diffraction, Resolution 2.65Å
Ligands:
Resources:FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

MTH1_HAEPH This methylase recognizes the double-stranded sequence GCGC, causes specific methylation on C-2 on both strands, and protects the DNA from cleavage by the HhaI endonuclease.

Evolutionary Conservation

Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.

See Also

2i9k, resolution 2.65Å

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