Nucleic Acids Research, 1993, Vol. 21, No. 15 3563-3566
© 1993
MOLECULAR BIOLOGY |
Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N6-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding
Department of Biology, University of Rochester Rochester, NY 14627, USA
To whom correspondence should be addressed
Received February 9, 1993. Revised June 11, 1993. Accepted June 11, 1993.
Comparison of the deduced amlno acid sequences of DNA-[N6-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY-motlf, or a variant of it. 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 (6 and 23-fold, respectively) Km for substrate, S-adenosyl-methlonlne (AdoMet) and an Increased (6 and 23-fold) K1 for product, S-adenosyl-homocysteine (AdoHcy); (ii) a slightly reduced (1.5 and 3-fold lower) kcat; (ill) a strongly reduced kcat/KmAdoMet (10 and 80-fold); and (iv) the same Km for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced (3 and 7-fold lower) Ka for AdoMet; all forms bound two molecules of 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-bindlng, and that region IV contains an AdoMet-binding site.