title: Enhanced macrocyclizing activity of the thioesterase from tyrocidine synthetase in presence of nonionic detergent.
abstract: Macrocyclization carried out by thioesterase domains of multimodular nonribosomal peptide synthetases (NRPSs) is a key step in the biosynthesis of many biologically active peptides. The thioesterase excised from tyrocidine synthetase is a versatile macrocyclization catalyst and a useful tool for chemoenzymatic synthesis of diverse cyclic peptides. However, its utility is limited by its short lifetime of catalytic activity as well as significant flux of the acyl-enzyme intermediate to hydrolysis. The addition of Brij 58, a nonionic detergent, above the critical micelle concentration, has dramatic effects on enzyme activity: catalytic activity is extended to >60 min and the rate of cyclization (but not hydrolysis) increases 6-fold, resulting in a net 150- to 300-fold increase in cyclic product yields. This enhanced activity allowed enzymatic macrocyclization of a solid phase library of tyrocidine decapeptides to identify acceptable substitutions at the Orn9 position which had previously been inaccessible for diversification.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Ellen
authors.initials: E
authors.lastname: Yeh
authors.forename: Hening
authors.initials: H
authors.lastname: Lin
authors.forename: Susan L
authors.initials: SL
authors.lastname: Clugston
authors.forename: Rahul M
authors.initials: RM
authors.lastname: Kohli
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D002592:Cetomacrogol / Q000494:pharmacology*; D003500:Cyclization; D003902:Detergents; D007700:Kinetics; D047028:Macrocyclic Compounds / Q000378:metabolism; D009952:Ornithine / Q000737:chemistry; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D013869:Thiolester Hydrolases / Q000378:metabolism*; D013997:Time Factors
chemical_list: D003902:Detergents; D047028:Macrocyclic Compounds; D002592:Cetomacrogol; D009952:Ornithine; D013869:Thiolester Hydrolases; D010453:Peptide Synthases; C035180:tyrocidine synthetase