PDBsum entry 5n4e

Hydrolase

PDB id: 5n4e

Contents

Protein chains

714 a.a.

30 a.a.

31 a.a.

Ligands

GOL

Waters ×939

PDB id:

5n4e

Name:

Hydrolase

Title:

Prolyl oligopeptidase b from galerina marginata bound to 35mer hydrolysis and macrocyclization substrate - h698a mutant

Structure:

Prolyl oligopeptidase. Chain: a, b. Engineered: yes. Mutation: yes. Alpha-amanitin proprotein. Chain: c, d. Engineered: yes

Source:

Galerina marginata. Organism_taxid: 109633. Gene: popb. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008. Synthetic: yes. Organism_taxid: 109633

Resolution:

2.90Å R-factor: 0.251 R-free: 0.295

Authors:

C.M.Czekster,S.A.Mcmahon,H.Ludewig,J.H.Naismith

Key ref:

C.M.Czekster et al. (2017). Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates. Nat Commun, 8, 1045. PubMed id: 29051530

Date:

10-Feb-17 Release date: 01-Nov-17

Protein chains

H2E7Q8  (POPB_GALM3) -  Dual function macrocyclase-peptidase POPB from Galerina marginata (strain CBS 339.88)

Seq: 730 a.a.

Struc: 714 a.a.*

Protein chain

A0A067SLB9  (AAMA1_GALM3) -  Alpha-amanitin proprotein 1 from Galerina marginata (strain CBS 339.88)

Seq: 35 a.a.

Struc: 30 a.a.

Protein chain

A0A067SLB9  (AAMA1_GALM3) -  Alpha-amanitin proprotein 1 from Galerina marginata (strain CBS 339.88)

Seq: 35 a.a.

Struc: 31 a.a.

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 2: Chains A, B: E.C.3.4.21.26 - prolyl oligopeptidase.
• Reaction: Hydrolysis of Pro-|-Xaa >> Ala-|-Xaa in oligopeptides.
• Enzyme class 3: Chains C, D: E.C.?

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Nat Commun 8:1045 (2017)

PubMed id: 29051530

Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates.

C.M.Czekster, H.Ludewig, S.A.McMahon, J.H.Naismith.

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.