All DNA methyltransferases (MTases) have similar catalytic domains containing nine blocks of conserved @entity535 residues. We have investigated by site-directed mutagenesis the function of 17 conserved residues in the EcoRV alpha- @entity5536 -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 @entity7726 ( @entity7726 ) cofactor. @entity1522 located in motifs X, I, and II are shown to be involved in @entity7726 binding (Lys16, @entity367 37, @entity815 39, and @entity2574 58). Some of the mutants defective in @entity7726 binding are also impaired in DNA binding, suggesting allosteric interactions between the @entity7726 and DNA binding site. @entity2574 78 (motif III), which was supposed to form a @entity3884 bond to the @entity7726 on the basis of the structure predictions, turned out not to be important for @entity7726 binding, suggesting that motif III has not been identified correctly. @entity69792 and @entity69793 , having mutations in the putative DNA binding domain, are unable to bind to DNA. Residues located in motifs IV, V, VI, and @entity797 are involved in catalysis ( @entity2574 193, Tyr196, @entity2574 211, 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 @entity7726 binding but strongly reduce catalysis. Our results are in substantial agreement with the structure prediction for EcoRV alpha- @entity5536 -N6-methyltransferase and x-ray structures of other MTases.

Functional roles of conserved @entity535 residues in DNA methyltransferases investigated by site-directed mutagenesis of the EcoRV XXXX -N6-methyltransferase.

@entity1522 :: ('CHEBI:33709', 'Chemical') :: ['Amino acids'] @entity3884 :: ('MESH:D006859', 'Chemical') :: ['hydrogen'] @entity535 :: ('CHEBI:33704', 'Chemical') :: ['amino acid'] @entity797 :: ('1351', 'Gene') :: ['VIII'] @entity5536 :: ('MESH:D000225', 'Chemical') :: ['adenine'] @entity7726 :: ('MESH:D012436', 'Chemical') :: ['S-adenosylmethionine', 'AdoMet'] @entity367 :: ('CHEBI:18237', 'Chemical') :: ['Glu'] @entity69792 :: ('p|SUB|R|128|A', 'ProteinMutation') :: ['R128A'] @entity815 :: ('CHEBI:28044', 'Chemical') :: ['Phe'] @entity2574 :: ('CHEBI:22660', 'Chemical') :: ['Asp'] @entity69793 :: ('p|SUB|N|130|A', 'ProteinMutation') :: ['N130A']
@entity5536 :: (MESH:D000225,Chemical) :: ['adenine']
