Photoaffinity labeling of the N-methyltransferase domains of cyclosporin synthetase.
The multifunctional polypeptide cyclosporin synthetase (CySyn) remains one of the most complex nonribosomal peptide synthetase described. In this study we used a highly specific photoaffinity labeling procedure with the natural cofactor S-adenosyl-L-methionine (AdoMet), 14C-isotopically labeled at the Sdelta methyl group to probe the concerted AdoMet-binding interaction of the N-methyltransferase (N-MTase) centers of CySyn. The binding stoichiometry for the enzyme-AdoMet complex was determined to be 1:7, which is in agreement with inferences made from analysis of the complementary DNA sequence of the simA gene encoding the CySyn polypeptide. The photolabeling of the AdoMet-binding sites displayed homotropic negative cooperativity, characterized by a curvilinear Scatchard plot with upward concavity. Although, the process of N-methyl transfer is not a critical event for peptide elongation, the destabilizing homotropic interactions between N-MTase centers that were observed may represent a mechanism whereby the enzyme preserves the proficiency of the substrate-channeling process of cyclosporin peptide assembly over a broad range of cofactor concentrations. Furthermore, we demonstrated the utility of the photolabeling procedure for tracking the enzyme during purification.
Velkov
Tony
T
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Victoria, Australia.
Lawen
Alfons
A
D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism; D019920:Photoaffinity Labels; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D009097:Multienzyme Complexes; D019920:Photoaffinity Labels; C048525:cyclosporin A synthetase; D010453:Peptide Synthases