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J. Biol. Chem., Vol. 279, Issue 50, 52075-52081, December 10, 2004

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Functional Characterization of Escherichia coli DNA Adenine Methyltransferase, a Novel Target for Antibiotics^*

Neda Mashhoon, Michael Carroll, Cynthia Pruss, Joerg Eberhard, Sawako Ishikawa, R. August Estabrook, and Norbert Reich¶||

From the EpiGenX Pharmaceuticals, Pacific Technology Center, Santa Barbara, California 93111, the Department of Operative Dentistry and Periodontology, University of Kiel, Kiel, Germany, and the ^¶Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106

We have characterized Escherichia coli DNA adenine methyltransferase, a critical regulator of bacterial virulence. Steady-state kinetics, product inhibition, and isotope exchange studies are consistent with a kinetic mechanism in which the cofactor S-adenosylmethionine binds first, followed by sequence-specific DNA binding and catalysis. The enzyme has a fast methyl transfer step followed by slower product release steps, and we directly demonstrate the competence of the enzyme cofactor complex. Methylation of adjacent GATC sites is distributive with DNA derived from a genetic element that controls the transcription of the adjacent genes. This indicates that the first methylation event is followed by enzyme release. The affinity of the enzyme for both DNA and S-adenosylmethionine was determined. Our studies provide a basis for further structural and functional analysis of this important enzyme and for the identification of inhibitors for potential therapeutic applications.

Received for publication, July 20, 2004 , and in revised form, September 21, 2004.

^* This work was supported by National Science Foundation Grant MCB-9983125 (to N. R.) and National Institutes of Health Grant SBIR II 2 R43 AI48377-01A1 (to EpiGenX Pharmaceuticals).

^|| To whom correspondence should be addressed. Tel.: 805-893-8368; E-mail: reich{at}chem.ucsb.edu.

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