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J. Biol. Chem., Vol. 275, Issue 40, 30826-30832, October 6, 2000

This Article Full Text Full Text (PDF) All Versions of this Article: 275/40/30826    most recent M002614200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hacker, C. Articles by Zocher, R. Search for Related Content PubMed PubMed Citation Articles by Hacker, C. Articles by Zocher, R. Social Bookmarking                      What's this?

Mutational Analysis of the N-Methyltransferase Domain of the Multifunctional Enzyme Enniatin Synthetase^*

Christine Hacker, Mirko Glinski, Till Hornbogen, Anke Doller, and Rainer Zocher^§

From the Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Fachgebiet Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstrasse 29, D-10587 Berlin, Germany

N-Methylcyclopeptides like cyclosporins and enniatins are synthesized by multifunctional enzymes representing hybrid systems of peptide synthetases and S-adenosyl-L-methionine (AdoMet)-dependent N-methyltransferases. The latter constitute a new family of N-methyltransferases sharing high homology within procaryotes and eucaryotes. Here we describe the mutational analysis of the N-methyltransferase domain of enniatin synthetase from Fusarium scirpi to gain insight into the assembly of the AdoMet-binding site. The role of four conserved motifs (I, ²⁰⁸⁵VLEIGTGSGMIL; II/Y, ²¹⁰⁵SYVGLDPS; IV, ²¹⁵²DLVVFNSVVQYFTPPEYL; and V, ²¹⁹⁴ATNGHFLAARA) in cofactor binding as measured by photolabeling was studied. Deletion of the first 21 N-terminal amino acid residues of the N-methyltransferase domain did not affect AdoMet binding. Further shortening close to motif I resulted in loss of binding activity. Truncation of 38 amino acids from the C terminus and also internal deletions containing motif V led to complete loss of AdoMet-binding activity. Point mutations converting the conserved Tyr²²³ (corresponding to position 2106 in enniatin synthetase) in motif II/Y (close to motif I) into Val, Ala, and Ser, respectively, strongly diminished AdoMet binding, whereas conversion of this residue to Phe restored AdoMet-binding activity to ~70%, indicating that Tyr²²³ is important for AdoMet binding and that the aromatic Tyr²²³ may be crucial for AdoMet binding in N-methylpeptide synthetases.

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