(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Eukaryota: NE > Viridiplantae: NE > Streptophyta: NE > Streptophytina: NE > Embryophyta: NE > Tracheophyta: NE > Euphyllophyta: NE > Spermatophyta: NE > Magnoliophyta: NE > Mesangiospermae: NE > eudicotyledons: NE > Gunneridae: NE > Pentapetalae: NE > Caryophyllales: NE > Caryophyllaceae: NE > Caryophylleae: NE > Gypsophila: NE > Gypsophila vaccaria: NE
Molecular evidence
Database
No mutation 9 structures(e.g. : 5O3U, 5O3V, 5O3W... more)(less) 5O3U: Structural characterization of the fast and promiscuous macrocyclase from plant - PCY1-S562A bound to Presegetalin F1, 5O3V: Structural characterization of the fast and promiscuous macrocyclase from plant Gypsophila vaccaria (Cow soapwort) - PCY1-S562A bound to Presegetalin B1, 5O3W: Structural characterization of the fast and promiscuous macrocyclase from plant Gypsophila vaccaria (Cow soapwort) - PCY1-S562A bound to Presegetalin A1, 5O3X: Structural characterization of the fast and promiscuous macrocyclase from plant Gypsophila vaccaria (Cow soapwort) - apo PCY1, 5UW3: Gypsophila vaccaria (Cow soapwort) macrocyclase PCY1 involved in the biosynthesis of RiPP cyclic peptide in complex with follower peptide, 5UW5: Gypsophila vaccaria (Cow soapwort) macrocyclase PCY1 involved in the biosynthesis of RiPP cyclic peptide PCY1 H695A Variant in Complex with Follower Peptide, 5UW6: Gypsophila vaccaria (Cow soapwort) macrocyclase PCY1 involved in the biosynthesis of RiPP cyclic peptide in complex with follower peptide and covalent inhibitor ZPP, 5UW7: PCY1 Y481F Variant in Complex with Follower Peptidee, 5UZW: PCY1 G696Insertion Variant in Complex with Follower Peptide and the Covalent Inhibitor ZPP Z-Pro-Prolinal No kinetic
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MATSGFSKPLHYPPVRRDETVVDDYFGVKVADPYRWLEDPNSEETKEFVD NQEKLANSVLEECELIDKFKQKIIDFVNFPRCGVPFRRANKYFHFYNSGL QAQNVFQMQDDLDGKPEVLYDPNLREGGRSGLSLYSVSEDAKYFAFGIHS GLTEWVTIKILKTEDRSYLPDTLEWVKFSPAIWTHDNKGFFYCPYPPLKE GEDHMTRSAVNQEARYHFLGTDQSEDILLWRDLENPAHHLKCQITDDGKY FLLYILDGCDDANKVYCLDLTKLPNGLESFRGREDSAPFMKLIDSFDASY TAIANDGSVFTFQTNKDAPRKKLVRVDLNNPSVWTDLVPESKKDLLESAH AVNENQLILRYLSDVKHVLEIRDLESGALQHRLPIDIGSVDGITARRRDS VVFFKFTSILTPGIVYQCDLKNDPTQLKIFRESVVPDFDRSEFEVKQVFV PSKDGTKIPIFIAARKGISLDGSHPCEMHGYGGFGINMMPTFSASRIVFL KHLGGVFCLANIRGGGEYGEEWHKAGFRDKKQNVFDDFISAAEYLISSGY TKARRVAIEGGSNGGLLVAACINQRPDLFGCAEANCGVMDMLRFHKFTLG YLWTGDYGCSDKEEEFKWLIKYSPIHNVRRPWEQPGNEETQYPATMILTA DHDDRVVPLHSFKLLATMQHVLCTSLEDSPQKNPIIARIQRKAAHYGRAT MTQIAEVADRYGFMAKALEAPWID
Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1's potential in biocatalysis.
Title: Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants Chekan JR, Estrada P, Covello PS, Nair SK Ref: Proc Natl Acad Sci U S A, 114:6551, 2017 : PubMed
Enzymes that can catalyze the macrocyclization of linear peptide substrates have long been sought for the production of libraries of structurally diverse scaffolds via combinatorial gene assembly as well as to afford rapid in vivo screening methods. Orbitides are plant ribosomally synthesized and posttranslationally modified peptides (RiPPs) of various sizes and topologies, several of which are shown to be biologically active. The diversity in size and sequence of orbitides suggests that the corresponding macrocyclases may be ideal catalysts for production of cyclic peptides. Here we present the biochemical characterization and crystal structures of the plant enzyme PCY1 involved in orbitide macrocyclization. These studies demonstrate how the PCY1 S9A protease fold has been adapted for transamidation, rather than hydrolysis, of acyl-enzyme intermediates to yield cyclic products. Notably, PCY1 uses an unusual strategy in which the cleaved C-terminal follower peptide from the substrate stabilizes the enzyme in a productive conformation to facilitate macrocyclization of the N-terminal fragment. The broad substrate tolerance of PCY1 can be exploited as a biotechnological tool to generate structurally diverse arrays of macrocycles, including those with nonproteinogenic elements.
Title: The two-step biosynthesis of cyclic peptides from linear precursors in a member of the plant family Caryophyllaceae involves cyclization by a serine protease-like enzyme Barber CJ, Pujara PT, Reed DW, Chiwocha S, Zhang H, Covello PS Ref: Journal of Biological Chemistry, 288:12500, 2013 : PubMed
Caryophyllaceae-type cyclic peptides (CPs) of 5-12 proteinogenic amino acids occur in 10 plant families. In Saponaria vaccaria (Caryophyllaceae), they have been shown to be formed from linear peptide precursors derived from ribosomal translation. There is also evidence for such precursors in other members of the Caryophyllaceae, Rutaceae, and Linaceae families. The biosynthesis of CP in the developing seeds of S. vaccaria was investigated with respect to the enzymes involved in precursor processing. Through biochemical assays with seed extracts and synthetic peptides, an enzyme named oligopeptidase 1 (OLP1) was found that catalyzes the cleavage of intermediates at the N terminus of the incipient CP. A second enzyme, peptide cyclase 1 (PCY1), which was separated chromatographically from OLP1, was found to act on the product of OLP1, giving rise to a cyclic peptide and concomitant removal of a C-terminal flanking sequence. PCY1 was partially purified, and using the methods of proteomics, a full-length cDNA clone encoding an enzyme matching the properties of PCY1 was obtained. The substrate specificity of purified recombinant PCY1, believed to be the first cloned plant enzyme whose function is peptide cyclization, was tested with synthetic peptides. The results are discussed in the light of CP biosynthetic systems of other organisms.
Gypsophila vaccaria Prolyl endopeptidase Pcy1
