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| | {{STRUCTURE_2hr1| PDB=2hr1 | SCENE= }} | | {{STRUCTURE_2hr1| PDB=2hr1 | SCENE= }} |
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| - | '''Ternary structure of WT M.HhaI C5-Cytosine DNA methyltransferase with unmodified DNA and AdoHcy'''
| + | ===Ternary structure of WT M.HhaI C5-Cytosine DNA methyltransferase with unmodified DNA and AdoHcy=== |
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| - | ==Overview==
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| - | Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased approximately 16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 10(5)-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 A resolution) shows the target cytosine to be positioned approximately 30 degrees into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to approximately +171 degrees, from a range of -95 degrees to -120 degrees in B DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in contrast to the previously reported C3'-endo (North conformation) described for the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 A) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4'-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_16926025}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 16926025 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_16926025}} |
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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: High resolution]] | | [[Category: High resolution]] |
| | [[Category: M hhai]] | | [[Category: M hhai]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun May 4 06:36:31 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jul 28 12:20:16 2008'' |
Revision as of 09:20, 28 July 2008
Template:STRUCTURE 2hr1
Ternary structure of WT M.HhaI C5-Cytosine DNA methyltransferase with unmodified DNA and AdoHcy
Template:ABSTRACT PUBMED 16926025
About this Structure
2HR1 is a Single protein structure of sequence from Haemophilus parahaemolyticus. Full crystallographic information is available from OCA.
Reference
The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI., Shieh FK, Youngblood B, Reich NO, J Mol Biol. 2006 Sep 22;362(3):516-27. Epub 2006 Jul 22. PMID:16926025
Page seeded by OCA on Mon Jul 28 12:20:16 2008
