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Nucleic Acids Res. 1993 Oct 11;21(20):4659-62.

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

Author information

1
Department of Biology, University of Rochester, NY 14627.

Abstract

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.

Corrected and republished from

  • Nucleic Acids Res 1993 Jul 25;21(15):3563-6.
PMID:
8233814
PMCID:
PMC331487
DOI:
10.1093/nar/21.20.4659
[Indexed for MEDLINE]
Free PMC Article

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