2i9k
From Proteopedia
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| - | [[Image:2i9k.gif|left|200px]] | + | [[Image:2i9k.gif|left|200px]] |
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| - | '''Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI''' | + | {{Structure |
| + | |PDB= 2i9k |SIZE=350|CAPTION= <scene name='initialview01'>2i9k</scene>, resolution 2.65Å | ||
| + | |SITE= | ||
| + | |LIGAND= <scene name='pdbligand=SAH:S-ADENOSYL-L-HOMOCYSTEINE'>SAH</scene> | ||
| + | |ACTIVITY= | ||
| + | |GENE= hhaIM ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=726 Haemophilus haemolyticus]) | ||
| + | }} | ||
| + | |||
| + | '''Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI''' | ||
| + | |||
==Overview== | ==Overview== | ||
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==About this Structure== | ==About this Structure== | ||
| - | 2I9K is a [ | + | 2I9K is a [[Single protein]] structure of sequence from [http://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]. |
==Reference== | ==Reference== | ||
| - | 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:[http:// | + | 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:[http://www.ncbi.nlm.nih.gov/pubmed/16919299 16919299] |
[[Category: Haemophilus haemolyticus]] | [[Category: Haemophilus haemolyticus]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
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[[Category: phe124ala mutation in m hhai]] | [[Category: phe124ala mutation in m hhai]] | ||
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Mar 20 17:26:53 2008'' |
Revision as of 15:26, 20 March 2008
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| , resolution 2.65Å | |||||||
|---|---|---|---|---|---|---|---|
| Ligands: | |||||||
| Gene: | hhaIM (Haemophilus haemolyticus) | ||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||
Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI
Overview
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.
About this Structure
2I9K is a Single protein structure of sequence from Haemophilus haemolyticus. Full crystallographic information is available from OCA.
Reference
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
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