title: Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase.
abstract: Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Harris
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Durek
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Quentin
authors.initials: Q
authors.lastname: Kaas
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Aaron G
authors.initials: AG
authors.lastname: Poth
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Edward K
authors.initials: EK
authors.lastname: Gilding
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
authors.forename: Brendon F
authors.initials: BF
authors.lastname: Conlan
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Ivana
authors.initials: I
authors.lastname: Saska
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Norelle L
authors.initials: NL
authors.lastname: Daly
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
authors.forename: Nicole L
authors.initials: NL
authors.lastname: van der Weerden
authors.affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.affiliation: Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
authors.forename: Marilyn A
authors.initials: MA
authors.lastname: Anderson
mesh_terms: D003500:Cyclization; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D032067:Oldenlandia; D010455:Peptides / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase
chemical_list: D003546:Cysteine Endopeptidases; D010455:Peptides; D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000099240:Asparaginyl Endopeptidase