Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N6-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding.

Comparison of the deduced amino acid sequences of DNA-N6-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY or closely related] motif. By site-directed mutagenesis of a cloned T4 dam gene, we have altered the first proline residue in this motif located in conserved region IV of the T4 Dam-MTase] to alanine or threonine. The mutant enzymic forms, P172A and P172T, were overproduced and purified. Kinetic studies showed that compared to the wild-type wt] the two mutant enzymic forms had: (i) an increased 5 and 20-fold, respectively] Km for substrate, S-adenosyl-methionine AdoMet]; (ii) a slightly reduced 2 and 4-fold lower] kcat; (iii) a strongly reduced kcat/KmAdoMet 10 and 100-fold]; and (iv) almost the same Km for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced 4 and 9-fold lower] Ka for AdoMet. Taken together these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPPY-motif is important for AdoMet-binding, and that region IV contains or is part of an AdoMet-binding site.