Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates

Abstract

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.

Molecular Basis for Autocatalytic Backbone N-Methylation in RiPP Natural Product Biosynthesis

“…The enzyme most likely possesses the follower peptide in order to serve as a recognition motif for the prolyl oligopeptidase that subsequently cyclizes the methylated product, as is the case for GmPOPB in amanitin biosynthesis, 32 and PCY1 in orbitide biosynthesis. 33, 34…”

Asparaginyl endopeptidases: enzymology, applications and limitations

“…The use of peptide ligases offers an alternative approach to peptide cyclisation. With excellent reaction kinetics and a short recognition sequence that is orthogonal to the existing enzymes such as the prolyl oligopeptidase, PCY1, 70 and transpeptidase, sortase A, 63,71 the addition of AEPs complements existing strategies.…”

Exploring the conformational landscape of a lanthipeptide synthetase using native mass spectrometry

“…rapidly expanding with the discovery and characterization of many new biosynthetic pathways, elucidation of the structural features that endow these enzymes with their relaxed substrate specificities and interesting catalytic properties have lagged behind. Nevertheless, high-resolution structures have been solved for a handful of enzymes involved in the biosynthesis of several types of RiPP natural products including the class I and II lanthipeptides, [6][7][8][9][10] cyanobactins, [11][12] orbitides, 13 sactipeptides, 14 lasso peptides, 15 thiopeptides, 16 and others. [17][18][19] In several systems, co-crystal structures of the RiPP biosynthetic enzyme bound to its cognate precursor peptide have revealed the presence of a winged helix-turn-helix motif (termed the RiPP recognition element 20 Recently, we reported the use hydrogen-deuterium exchange mass spectrometry (HDX-MS) as a versatile biophysical tool for studying the structural dynamics of RiPP biosynthetic enzymes.…”