title: Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase.
abstract: We employed a highly specific photoaffinity labeling procedure, using (14)C-labeled S-adenosyl-l-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-(14)C]AdoMet or [carboxyl-(14)C]AdoMet yielded the sequence H(2)N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu(10654) and Pro(10655) as the major sites of derivatization. [carboxyl-(14)C]AdoMet in addition labeled Tyr(10650). Chymotryptic digestion generated the radiolabeled peptide H(2)N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu(2128) and Pro(2129), which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Building 13D, Melbourne, Victoria 3800, Australia.
authors.forename: Tony
authors.initials: T
authors.lastname: Velkov
authors.forename: Alfons
authors.initials: A
authors.lastname: Lawen
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002855:Chromatography, Thin Layer; D008780:Methyltransferases / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D010453:Peptide Synthases / Q000737:chemistry*; D011487:Protein Conformation; D012436:S-Adenosylmethionine / Q000737:chemistry*
chemical_list: D009097:Multienzyme Complexes; C048525:cyclosporin A synthetase; D012436:S-Adenosylmethionine; D008780:Methyltransferases; D010453:Peptide Synthases