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1.  Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N6-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding 
Nucleic Acids Research  1993;21(15):3563-3566.
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.
PMCID: PMC331459  PMID: 16617501
3.  A DNA Adenine Methyltransferase of Escherichia coli That Is Cell Cycle Regulated and Essential for Viability 
Journal of Bacteriology  2004;186(7):2061-2067.
DNA sequence analysis revealed that the putative yhdJ DNA methyltransferase gene of Escherichia coli is 55% identical to the Nostoc sp. strain PCC7120 gene encoding DNA methyltransferase AvaIII, which methylates adenine in the recognition sequence, ATGCAT. The yhdJ gene was cloned, and the enzyme was overexpressed and purified. Methylation and restriction analysis showed that the DNA methyltransferase methylates the first adenine in the sequence ATGCAT. This DNA methylation was found to be regulated during the cell cycle, and the DNA adenine methyltransferase was designated M.EcoKCcrM (for “cell cycle-regulated methyltransferase”). The CcrM DNA adenine methyltransferase is required for viability in E. coli, as a strain lacking a functional genomic copy of ccrM can be isolated only in the presence of an additional copy of ccrM supplied in trans. The cells of such a knockout strain stopped growing when expression of the inducible plasmid ccrM gene was shut off. Overexpression of M.EcoKCcrM slowed bacterial growth, and the ATGCAT sites became fully methylated throughout the cell cycle; a high proportion of cells with an anomalous size distribution and DNA content was found in this population. Thus, the temporal control of this methyltransferase may contribute to accurate cell cycle control of cell division and cellular morphology. Homologs of M.EcoKCcrM are present in other bacteria belonging to the gamma subdivision of the class Proteobacteria, suggesting that methylation at ATGCAT sites may have similar functions in other members of this group.
PMCID: PMC374390  PMID: 15028690

Results 1-3 (3)