Functional roles of conserved amino acid residues in DNA methyltransferases investigated by site-directed mutagenesis of the EcoRV adenine-N6-methyltransferase.
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 alpha-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 alpha-adenine-N6-methyltransferase and x-ray structures of other MTases.
Roth
M
M
Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany.
Helm-Kruse
S
S
Friedrich
T
T
Jeltsch
A
A
D000595:Amino Acid Sequence; D002384:Catalysis; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012436:S-Adenosylmethionine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D012436:S-Adenosylmethionine; C056127:DNA modification methylase EcoRV; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)