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Kinetic and catalytic properties of dimeric KpnI DNA methyltransferase
Shivakumara Bheemanaik1, Siddamadappa Chandrashekaran, Valakunja Nagaraja
1Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Abstract:
KpnI DNA-(N(6)-adenine)-methyltransferase (KpnI MTase) is a member of a restriction-modification (R-M) system in Klebsiella pneumoniae and recognizes the sequence 5'-GGTACC-3'. It modifies the recognition sequence by transferring the methyl group from S-adenosyl-l-methionine (AdoMet) to the N(6) position of adenine residue. KpnI MTase occurs as a dimer in solution as shown by gel filtration and chemical cross-linking analysis. The nonlinear dependence of methylation activity on enzyme concentration indicates that the functionally active form of the enzyme is also a dimer. Product inhibition studies with KpnI MTase showed that S-adenosyl-l-homocysteine is a competitive inhibitor with respect to AdoMet and noncompetitive inhibitor with respect to DNA. The methylated DNA showed noncompetitive inhibition with respect to both DNA and AdoMet. A reduction in the rate of methylation was observed at high concentrations of duplex DNA. The kinetic analysis where AdoMet binds first followed by DNA, supports an ordered bi bi mechanism. After methyl transfer, methylated DNA dissociates followed by S-adenosyl-l-homocysteine. Isotope-partitioning analysis showed that KpnI MTase-AdoMet complex is catalytically active.
Insights
KpnI DNA-(N(6)-adenine)-methyltransferase (KpnI MTase) functions as a dimer in Klebsiella pneumoniae. Kinetic studies reveal an ordered bi bi mechanism for DNA methylation by KpnI MTase.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- KpnI DNA-(N(6)-adenine)-methyltransferase (KpnI MTase) is part of a restriction-modification system in Klebsiella pneumoniae.
- It recognizes and methylates the DNA sequence 5'-GGTACC-3' using S-adenosyl-l-methionine (AdoMet).
Purpose of the Study:
- To elucidate the oligomeric state and kinetic mechanism of KpnI MTase.
- To characterize the enzyme's interaction with its substrate and products.
Main Methods:
- Gel filtration and chemical cross-linking to determine enzyme oligomerization.
- Enzyme kinetics, including product inhibition and isotope-partitioning studies.
- Analysis of substrate and product inhibition patterns.
Main Results:
- KpnI MTase exists and functions as a dimer in solution.
- Kinetic analysis supports an ordered bi bi mechanism with AdoMet binding first, followed by DNA.
- S-adenosyl-l-homocysteine is a competitive inhibitor with respect to AdoMet, while methylated DNA shows noncompetitive inhibition.
- High duplex DNA concentrations reduce methylation rates.
- The KpnI MTase-AdoMet complex is catalytically active.
Conclusions:
- The dimeric form of KpnI MTase is the active species.
- The enzyme follows an ordered sequential kinetic mechanism for DNA methylation.
- Understanding these properties is crucial for studying restriction-modification systems and DNA modification enzymes.