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Kinetics of Methylation by EcoP1I DNA Methyltransferase
Shivakumara Bheemanaik1, Srivani Sistla, Vinita Krishnamurthy
1Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Abstract:
EcoP1I DNA MTase (M.EcoP1I), an N(6)-adenine MTase from bacteriophage P1, is a part of the EcoP1I restriction-modification (R-M) system which belongs to the Type III R-M system. It recognizes the sequence 5'-AGACC-3' and methylates the internal adenine. M.EcoP1I requires Mg(2+) for the transfer of methyl groups to DNA. M.EcoP1I is shown to exist as dimer in solution, and even at high salt concentrations (0.5 M) the dimeric M.EcoP1I does not dissociate into monomers suggesting a strong interaction between the monomer subunits. Preincubation and isotope partitioning studies with M.EcoP1I indicate a kinetic mechanism where the duplex DNA binds first followed by AdoMet. Interestingly, M.EcoP1I methylates DNA substrates in the presence of Mn(2+) and Ca(2+) other than Mg(2+) with varying affinities. Amino acid analysis and methylation assays in the presence of metal ions suggest that M.EcoP1I has indeed two metal ion-binding sites [(358)ID(x)(n) … ExK(401) and (600)DxDxD(604) motif]. EcoP1I DNA MTase catalyzes the transfer of methyl groups using a distributive mode of methylation on DNA containing more than one recognition site. A chemical modification of EcoP1I DNA MTase using N-ethylmaleimide resulted in an irreversible inactivation of enzyme activity suggesting the possible role of cysteine residues in catalysis.
Insights
EcoP1I DNA MTase, a Type III restriction-modification enzyme, methylates DNA at the 5'-AGACC-3' sequence. It functions as a dimer and exhibits distributive methylation, with potential roles for cysteine residues in catalysis.
Area of Science:
- Molecular Biology
- Enzymology
- Epigenetics
Background:
- EcoP1I DNA MTase (M.EcoP1I) is an N(6)-adenine methyltransferase from bacteriophage P1, part of the Type III restriction-modification system.
- It recognizes the 5'-AGACC-3' sequence and methylates the internal adenine, requiring Mg(2+) for activity.
Purpose of the Study:
- To elucidate the biochemical and mechanistic properties of M.EcoP1I.
- To identify metal ion-binding sites and understand the enzyme's kinetic mechanism and methylation mode.
Main Methods:
- Enzyme kinetics studies including preincubation and isotope partitioning.
- Methylation assays in the presence of various divalent metal ions (Mg(2+), Mn(2+), Ca(2+)).
- Amino acid analysis and chemical modification with N-ethylmaleimide.
Main Results:
- M.EcoP1I exists as a stable dimer in solution, even at high salt concentrations.
- The enzyme exhibits distributive methylation on DNA with multiple recognition sites.
- M.EcoP1I can utilize Mn(2+) and Ca(2+) in addition to Mg(2+), suggesting two metal-binding sites.
- Chemical modification indicated a potential role for cysteine residues in catalysis.
Conclusions:
- M.EcoP1I possesses a strong dimeric structure and a distributive methylation mechanism.
- The enzyme's active site accommodates multiple metal ions, and cysteine residues may be crucial for its catalytic function.
