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J Biol Chem, Vol. 273, Issue 28, 17333-17342, July 10, 1998
From the Institut für Biochemie, Fachbereich Biologie,
Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany
All DNA methyltransferases (MTases) have similar
catalytic domains containing nine blocks of conserved amino acid
residues. We have investigated by site-directed mutagenesis the
function of 17 conserved residues in the EcoRV
Functional Roles of Conserved Amino Acid Residues in DNA
Methyltransferases Investigated by Site-directed Mutagenesis of the
EcoRV
Adenine-N6-methyltransferase
-adenine-N6-DNA methyltransferase.
The structure of this class of MTases has been predicted recently. The
variants were characterized with respect to their catalytic activities
and their abilities to bind to DNA and the
S-adenosylmethionine (AdoMet) cofactor. Amino acids located
in motifs X, I, and II are shown to be involved in AdoMet binding
(Lys16, Glu37, Phe39, and
Asp58). Some of the mutants defective in AdoMet binding are
also impaired in DNA binding, suggesting allosteric interactions
between the AdoMet and DNA binding site. Asp78 (motif III),
which was supposed to form a hydrogen bond to the AdoMet on the basis
of the structure predictions, turned out not to be important for AdoMet
binding, suggesting that motif III has not been identified correctly.
R128A and N130A, having mutations in the putative DNA binding domain,
are unable to bind to DNA. Residues located in motifs IV, V, VI, and
VIII are involved in catalysis (Asp193, Tyr196,
Asp211, Ser229, Trp231, and
Tyr258), some of them presumably in binding the flipped
target base, because mutations at these residues fail to significantly
interfere with DNA and AdoMet binding but strongly reduce catalysis.
Our results are in substantial agreement with the structure prediction for EcoRV
-adenine-N6-methyltransferase and
x-ray structures of other MTases.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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