Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N6-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding
AUTOR(ES)
Kossykh, Valeri G.
RESUMO
Comparison of the deduced amino acid sequences of DNA-[N6-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY-motif, 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-methionine (AdoMet) and an increased (6 and 23-fold) Ki for product, S-adenosyl-homocysteine (AdoHcy); (ii) a slightly reduced (1.5 and 3-fold lower) kcat; (iii) 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-binding, and that region IV contains an AdoMet-binding site.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=331459Documentos Relacionados
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