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

  • Shivakumara Bheemanaik , Janusz M. Bujnicki , Valakunja Nagaraja und Desirazu N. Rao
Veröffentlicht/Copyright: 1. Juni 2006
Biological Chemistry
Aus der Zeitschrift Band 387 Heft 5

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.

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

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Published Online: 2006-06-01
Published in Print: 2006-05-01

©2006 by Walter de Gruyter Berlin New York

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https://doi.org/10.1515/BC.2006.067
Schlagwörter für diesen Artikel
DNA methyltransferase; KpnI DNA methyltransferase; S-adenosyl-l-methionine; target recognition domain

Artikel in diesem Heft

  1. Protein aggregation in crowded environments
  2. Nitrite, a naturally occurring precursor of nitric oxide that acts like a ‘prodrug’
  3. Functional studies of the small subunit of EcoHK31I DNA methyltransferase
  4. Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase
  5. Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific β recombinase, and identification of a folding intermediate
  6. Tyr-48, a conserved residue in ribotoxins, is involved in the RNA-degrading activity of α-sarcin
  7. Pathogenicity of catalytic antibodies: catalytic activity of Bence Jones proteins from myeloma patients with renal impairment can elicit cytotoxic effects
  8. Transgenic expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco
  9. Catalytic pathways of Euphorbia characias peroxidase reacting with hydrogen peroxide
  10. Biochemical and pharmacological characterization of the human bradykinin subtype 2 receptor produced in mammalian cells using the Semliki Forest virus system
  11. A spectroscopic analysis of the interaction between the human regulatory proteins RACK1 and Ki-1/57
  12. Subcellular localisation of human inositol 1,4,5-trisphosphate 3-kinase C: species-specific use of alternative export sites for nucleo-cytoplasmic shuttling indicates divergent roles of the catalytic and N-terminal domains
  13. The gating effect of calmodulin and calcium on the connexin50 hemichannel
  14. C-Terminal fusion of eGFP to the bradykinin B2 receptor strongly affects down-regulation but not receptor internalization or signaling
  15. Angiotensin I-converting enzyme inhibitor peptides derived from the endostatin-containing NC1 fragment of human collagen XVIII
  16. μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation
  17. Cathepsin L splice variants in human breast cell lines
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