Dam methyltransferase from Escherichia coli: sequence of a peptide segment involved in S-adenosyl-methionine binding.
DNA adenine methyltransferase (Dam methylase) has been crosslinked with its cofactor S-adenosyl methionine (AdoMet) by UV irradiation. About 3% of the enzyme was radioactively labelled after the crosslinking reaction performed either with (methyl-3H)-AdoMet or with (carboxy-14C)-AdoMet. Radiolabelled peptides were purified after trypsinolysis by high performance liquid chromatography in two steps. They could not be sequenced due to radiolysis. Therefore we performed the same experiment using non-radioactive AdoMet and were able to identify the peptide modified by the crosslinking reaction by comparison of the separation profiles obtained from two analytical control experiments performed with 3H-AdoMet and Dam methylase without crosslink, respectively. This approach was possible due to the high reproducibility of the chromatography profiles. In these three experiments only one radioactively labelled peptide was present in the tryptic digestions of the crosslinked enzyme. Its sequence was found to be XA-GGK, corresponding to amino acids 10-14 of Dam methylase. The non-identified amino acid in the first sequence cycle should be a tryptophan, which is presumably modified by the crosslinking reaction. The importance of this region near the N-terminus for the structure and function of the enzyme was also demonstrated by proteolysis and site-directed mutagenesis experiments.
Wenzel
C
C
Département de Biologie Cellulaire et Moléculaire, Centre d'Etudes de Saclay, Gif-sur-Yvette, France.
Guschlbauer
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W
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D012436:S-Adenosylmethionine / Q000378:metabolism*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D010455:Peptides; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499294:dam protein, E coli