title: Mutational analysis of the N-methyltransferase domain of the multifunctional enzyme enniatin synthetase.
abstract: 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, (2085)VLEIGTGSGMIL; II/Y, (2105)SYVGLDPS; IV, (2152)DLVVFNSVVQYFTPPEYL; and V, (2194)ATNGHFLAARA) 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 Tyr(223) (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 approximately 70%, indicating that Tyr(223) is important for AdoMet binding and that the aromatic Tyr(223) may be crucial for AdoMet binding in N-methylpeptide synthetases.
authors.affiliation: Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Fachgebiet Biochemie und Molekulare Biologie, Technische Universität Berlin, Franklinstrasse 29, D-10587 Berlin, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Hacker
authors.forename: M
authors.initials: M
authors.lastname: Glinski
authors.forename: T
authors.initials: T
authors.lastname: Hornbogen
authors.forename: A
authors.initials: A
authors.lastname: Doller
authors.forename: R
authors.initials: R
authors.lastname: Zocher
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005670:Fusarium / Q000201:enzymology; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D010453:Peptide Synthases; C023122:enniatin B synthetase