title: Semienzymatic cyclization of disulfide-rich peptides using Sortase A.
abstract: Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, α-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.
authors.affiliation: From QIMR Berghofer Medical Research, Brisbane 4000, Queensland, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia.
authors.forename: Xinying
authors.initials: X
authors.lastname: Jia
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia.
authors.forename: Soohyun
authors.initials: S
authors.lastname: Kwon
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia.
authors.forename: Ching-I Anderson
authors.initials: CA
authors.lastname: Wang
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia.
authors.forename: Yen-Hua
authors.initials: YH
authors.lastname: Huang
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia.
authors.forename: Lai Y
authors.initials: LY
authors.lastname: Chan
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia.
authors.forename: Chia Chia
authors.initials: CC
authors.lastname: Tan
authors.affiliation: School of Biomedical Sciences, University of Queensland, Brisbane 4072, Queensland, Australia.
authors.forename: K Johan
authors.initials: KJ
authors.lastname: Rosengren
authors.affiliation: From QIMR Berghofer Medical Research, Brisbane 4000, Queensland, Australia.
authors.forename: Jason P
authors.initials: JP
authors.lastname: Mulvenna
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia. Electronic address: c.schroeder@imb.uq.edu.au.
authors.forename: Christina I
authors.initials: CI
authors.lastname: Schroeder
authors.affiliation: Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Queensland, Australia. Electronic address: d.craik@imb.uq.edu.au.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D020916:Conotoxins / Q000737:chemistry; D003500:Cyclization; D047169:Cyclotides / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010456:Peptides, Cyclic / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D020916:Conotoxins; D047169:Cyclotides; D010455:Peptides; D010456:Peptides, Cyclic; C518080:acyclic sunflower trypsin inhibitor, Ser(3,11)-; C091930:kalata B1; C507383:alpha-conotoxin Vc1.1; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D003545:Cysteine
keywords: Cyclic Peptides; Cyclotides; Disulfide; Enzymatic Cyclization; Kalata B1; NMR; Peptide Chemical Synthesis; Peptides; Sortase A; Toxins