Ludewig H

References (2)

Title : Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates - Ludewig_2018_ACS.Chem.Biol_13_801
Author(s) : Ludewig H , Czekster CM , Oueis E , Munday ES , Arshad M , Synowsky SA , Bent AF , Naismith JH
Ref : ACS Chemical Biology , 13 :801 , 2018
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.
ESTHER : Ludewig_2018_ACS.Chem.Biol_13_801
PubMedSearch : Ludewig_2018_ACS.Chem.Biol_13_801
PubMedID: 29377663
Gene_locus related to this paper: 9cary-r4p353

Title : Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates - Czekster_2017_Nat.Commun_8_1045
Author(s) : Czekster CM , Ludewig H , McMahon SA , Naismith JH
Ref : Nat Commun , 8 :1045 , 2017
Abstract : Peptide macrocycles are promising therapeutic molecules because they are protease resistant, structurally rigid, membrane permeable, and capable of modulating protein-protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the 25 amino-acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino-acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal eight residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology.
ESTHER : Czekster_2017_Nat.Commun_8_1045
PubMedSearch : Czekster_2017_Nat.Commun_8_1045
PubMedID: 29051530
Gene_locus related to this paper: 9agar-h2e7q8