title: Peptide macrocyclization by a bifunctional endoprotease.
abstract: Proteases usually cleave peptides, but under some conditions, they can ligate them. Seeds of the common sunflower contain the 14-residue, backbone-macrocyclic peptide sunflower trypsin inhibitor 1 (SFTI-1) whose maturation from its precursor has a genetic requirement for asparaginyl endopeptidase (AEP). To provide more direct evidence, we developed an in situ assay and used (18)O-water to demonstrate that SFTI-1 is excised and simultaneously macrocyclized from its linear precursor. The reaction is inefficient in situ, but a newfound breakdown pathway can mask this inefficiency by reducing the internal disulfide bridge of any acyclic-SFTI to thiols before degrading it. To confirm AEP can directly perform the excision/ligation, we produced several recombinant plant AEPs in E. coli, and one from jack bean could catalyze both a typical cleavage reaction and cleavage-dependent, intramolecular transpeptidation to create SFTI-1. We propose that the evolution of ligating endoproteases enables plants like sunflower and jack bean to stabilize bioactive peptides.
authors.affiliation: The University of Western Australia, School of Chemistry and Biochemistry, 35 Stirling Highway, Crawley, Perth 6009, Australia; ARC Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley, Perth 6009, Australia.
authors.forename: Kalia
authors.initials: K
authors.lastname: Bernath-Levin
authors.affiliation: ARC Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley, Perth 6009, Australia.
authors.forename: Clark
authors.initials: C
authors.lastname: Nelson
authors.affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane 4072, Australia.
authors.forename: Alysha G
authors.initials: AG
authors.lastname: Elliott
authors.affiliation: The University of Western Australia, School of Chemistry and Biochemistry, 35 Stirling Highway, Crawley, Perth 6009, Australia; ARC Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley, Perth 6009, Australia.
authors.forename: Achala S
authors.initials: AS
authors.lastname: Jayasena
authors.affiliation: ARC Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley, Perth 6009, Australia.
authors.forename: A Harvey
authors.initials: AH
authors.lastname: Millar
authors.affiliation: The University of Queensland, Institute for Molecular Bioscience, Brisbane 4072, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.affiliation: The University of Western Australia, School of Chemistry and Biochemistry, 35 Stirling Highway, Crawley, Perth 6009, Australia; ARC Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley, Perth 6009, Australia. Electronic address: joshua.mylne@uwa.edu.au.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Mylne
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D032449:Canavalia / Q000201:enzymology; D003500:Cyclization; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006368:Helianthus / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes / Q000737:chemistry; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012639:Seeds / Q000378:metabolism; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry; D014867:Water / Q000737:chemistry; D000099240:Asparaginyl Endopeptidase
chemical_list: D003546:Cysteine Endopeptidases; D004220:Disulfides; D010103:Oxygen Isotopes; D010456:Peptides, Cyclic; D010940:Plant Proteins; D011994:Recombinant Proteins; D014867:Water; D000099240:Asparaginyl Endopeptidase; C448501:SFTI-1 peptide, sunflower