2i9k | pdb_00002i9k

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

spinqualitylabelsall models 2i9k, resolution 2.65Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 2i9k is a 3 chain structure with sequence from "bacillus_x"_pritchett_and_stillman_1919 "bacillus x" pritchett and stillman 1919. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: SAH Related: 2hr1, 3mht Gene: hhaIM ("Bacillus X" Pritchett and Stillman 1919) Activity: DNA (cytosine-5-)-methyltransferase, with EC number 2.1.1.37 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum 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. 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also DNA methyltransferase HhaI DNA methyltransferase

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