title: Investigation of the C-terminal domain of the bacterial DNA-(adenine N6)-methyltransferase CcrM.
abstract: CcrM-related DNA-(adenine N6)-methyltransferases play very important roles in the biology of Caulobacter crescentus and other alpha-proteobacteria. These enzymes methylate GANTC sequences, but the molecular mechanism by which they recognize their target sequence is unknown. We carried out multiple sequence alignments and noticed that CcrM enzymes contain a conserved C-terminal domain (CTD) which is not present in other DNA-(adenine N6)-methyltransferases and we show here that deletion of this part abrogates catalytic activity and DNA binding of CcrM. A mutational study identified 7 conserved residues in the CTD (out of 13 tested), mutation of which led to a strong reduction in catalytic activity. All of these mutants showed altered DNA binding, but no change in AdoMet binding and secondary structure. Some mutants exhibited reduced DNA binding, but others showed an enhanced DNA binding. Moreover, we show that CcrM does not specifically bind to DNA containing GANTC sequences. Taken together, these findings suggest that the specific CcrM-DNA complex undergoes a conformational change, which is endergonic but essential for catalytic activity and this step is blocked by some of the mutations. Moreover, our data indicate that the CTD of CcrM is involved in DNA binding and recognition. This suggests that the CTD functions as target recognition domain of CcrM and, therefore, CcrM can be considered the first example of a δ-type DNA-(adenine N6)-methyltransferase identified so far.
authors.affiliation: Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany.
authors.forename: Johannes A H
authors.initials: JA
authors.lastname: Maier
authors.affiliation: Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany.
authors.forename: Razvan F
authors.initials: RF
authors.lastname: Albu
authors.affiliation: Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany.
authors.forename: Tomasz P
authors.initials: TP
authors.lastname: Jurkowski
authors.affiliation: Institute of Biochemistry, Stuttgart University, 70569 Stuttgart, Germany. Electronic address: albert.jeltsch@ibc.uni-stuttgart.de.
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D016935:Caulobacter crescentus / Q000201:enzymology*; D018388:Codon, Terminator; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D019175:DNA Methylation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D018388:Codon, Terminator; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
keywords: CcrM; DNA methylation; DNA recognition; Molecular evolution