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 hyltransferases has revealed several conserved regions. All of these e nzymes contain a DPPY-motif, or a variant of it. By site-directed muta genesis of a cloned T4 dam gene, we have altered the first proline res idue 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 t o the wild-type (wt) the two mutant enzymic forms had: (i) an increase d (6 and 23-fold, respectively) K(m) for substrate, S-adenosyl-methion ine (AdoMet) and an increased (6 and 23-fold) K(i) for product, S-aden osyl-homocysteine (AdoHcy); (ii) a slightly reduced (1.5 and 3-fold lo wer) k(cat); (iii) a strongly reduced k(cat)/K(m)AdoMet (10 and 80-fol d); and (iv) the same K(m) for substrate DNA. Equilibrium dialysis stu dies showed that the mutant enzymes had a reduced (3 and 7-fold lower) K(a) for AdoMet; all forms bound two molecules of AdoMet. Taken toget her these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPP Y-motif is important for AdoMet-binding, and that region IV contains a n AdoMet-binding site.