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]-met hyltransterases has revealed several conserved regions. All of these e nzymes contain a DPPY [or closely related] motif. By site-directed mut agenesis of a cloned T4 dam gene, we have altered the first proline re sidue in this motif [located in conserved region IV of the T4 Dam-MTas e] 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 increas ed [5 and 20-fold, respectively] K(m) for substrate, S-adenosyl-methio nine [AdoMet]; (ii) a slightly reduced [2 and 4-fold lower] k(cat); (i ii) a strongly reduced k(cat)/K(m)AdoMet [10 and 100-fold]; and (iv) a lmost the same K(m) for substrate DNA. Equilibrium dialysis studies sh owed that the mutant enzymes had a reduced [4 and 9-fold lower] K(a) f or 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 tha t region IV contains or is part of an AdoMet-binding site.