Bheemanaik, Shivakumara ; Bujnicki, Janusz M. ; Nagaraja, Valakunja ; Rao, Desirazu N. (2006) Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase Biological Chemistry, 387 (5). pp. 515-523. ISSN 1431-6730
Full text not available from this repository.
Official URL: http://www.degruyter.com/view/j/bchm.2006.387.issu...
Related URL: http://dx.doi.org/10.1515/BC.2006.067
Abstract
KpnI DNA-(N6-adenine) methyltransferase (M.KpnI) recognises the sequence 5'-GGTACC-3' and transfers the methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of the adenine residue in each strand. Earlier studies have shown that M.KpnI exists as a dimer in solution, unlike most other MTases. To address the importance of dimerisation for enzyme function, a three-dimensional model of M.KpnI was obtained based on protein fold-recognition analysis, using the crystal structures of M.RsrI and M.MboIIA as templates. Residues I146, I161 and Y167, the side chains of which are present in the putative dimerisation interface in the model, were targeted for site-directed mutagenesis. Methylation and in vitro restriction assays showed that the mutant MTases are catalytically inactive. Mutation at the I146 position resulted in complete disruption of the dimer. The replacement of I146 led to drastically reduced DNA and cofactor binding. Substitution of I161 resulted in weakening of the interaction between monomers, leading to both monomeric and dimeric species. Steady-state fluorescence measurements showed that the wild-type KpnI MTase induces structural distortion in bound DNA, while the mutant MTases do not. The results establish that monomeric MTase is catalytically inactive and that dimerisation is an essential event for M.KpnI to catalyse the methyl transfer reaction.
Item Type: | Article |
---|---|
Source: | Copyright of this article belongs to Walter de Gruyter GmbH & Co. KG. |
Keywords: | DNA Methyltransferase; KpnI DNA Methyltransferase; S-adenosyl-l-methionine; Target Recognition Domain |
ID Code: | 89558 |
Deposited On: | 28 Apr 2012 14:30 |
Last Modified: | 05 Jul 2012 08:49 |
Repository Staff Only: item control page