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Nucleic Acids Research, 1993, Vol. 21, No. 19 4604-4609
© 1993

ENZYMOLOGY

Dam methyltransferase from Escherichia coLi: sequence of a peptide segment involved in S-adenosyl-methionine binding

Caroline Wenzel and Wilhelm Guschlbauer*

Service de Biochimie et Geéneétique Moleéculaire Ba^t. 142, Deépartement de Biologie Cellulaire et Moleéculaire Centre d'Etudes de Saclay F-91191 Gif-sur-Yvette Cedex, France

*To whom correspondence should be addressed

Received May 13, 1993. Revised August 26, 1993. Accepted August 26, 1993.

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 3HAdoMet 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 XAGGK, 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.


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