Functional analysis of amino acid residues at the dimerisation interface of KpnI DNA methyltransferase.
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.
Bheemanaik
Shivakumara
S
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Bujnicki
Janusz M
JM
Nagaraja
Valakunja
V
Rao
Desirazu N
DN
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000596:Amino Acids; D021122:Protein Subunits; C072049:DNA modification methylase KpnI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)