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Originally published In Press as doi:10.1074/jbc.M211458200 on December 28, 2002

J. Biol. Chem., Vol. 278, Issue 10, 7863-7874, March 7, 2003
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Kinetic and Catalytic Properties of Dimeric KpnI DNA Methyltransferase*

Shivakumara BheemanaikDagger , Siddamadappa Chandrashekaran§, Valakunja Nagaraja§, and Desirazu N. RaoDagger

From the Dagger  Department of Biochemistry, and § Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India

KpnI DNA-(N6-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 N6 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.

* This work was supported by a grant from Department of Science and Technology, Government of India.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 91-80-3942538; Fax: 91-80-3600814; E-mail: dnrao@biochem.iisc.ernet.in.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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