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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board Member: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Sies, Helmut / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred

12 Issues per year


IMPACT FACTOR 2016: 3.273

CiteScore 2016: 3.01

SCImago Journal Rank (SJR) 2016: 1.679
Source Normalized Impact per Paper (SNIP) 2016: 0.800

Online
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1437-4315
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Volume 387, Issue 5 (May 2006)

Issues

Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase

Shivakumara Bheemanaik
  • Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
/ Janusz M. Bujnicki
  • Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland
/ Valakunja Nagaraja
  • Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India
/ Desirazu N. Rao
  • Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India
Published Online: 2006-06-01 | DOI: https://doi.org/10.1515/BC.2006.067

Abstract

KpnI DNA-(N 6-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.

Keywords: DNA methyltransferase; KpnI DNA methyltransferase; S-adenosyl-l-methionine; target recognition domain

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Corresponding author


Received: December 14, 2005

Accepted: February 24, 2006

Published Online: 2006-06-01

Published in Print: 2006-05-01


Citation Information: Biological Chemistry, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/BC.2006.067.

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