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1 February 2003 Photoaffinity Labeling of the N-methyltransferase Domains of Cyclosporin Synthetase
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Abstract

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 Sδ 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.

Tony Velkov and Alfons Lawen "Photoaffinity Labeling of the N-methyltransferase Domains of Cyclosporin Synthetase," Photochemistry and Photobiology 77(2), 129-137, (1 February 2003). https://doi.org/10.1562/0031-8655(2003)077<0129:PLOTNM>2.0.CO;2
Received: 11 July 2002; Accepted: 1 November 2002; Published: 1 February 2003
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