PDBsum entry 4cth

Transferase

PDB id: 4cth

Contents

Protein chain

697 a.a.

Ligands

NAG ×3

GOL ×8

RDF

Metals

_ZN

Waters ×325

PDB id:

4cth

Name:

Transferase

Title:

Neprilysin variant g399v,g714k in complex with phosphoramidon

Structure:

Neprilysin. Chain: a. Fragment: extracellular domain, residues 52-750. Synonym: atriopeptidase, common acute lymphocytic leukemia antigen, calla, enkephalinase, neutral endopeptidase 24.11, nep, neutral endopeptidase, skin fibroblast elastase, sfe, cd10. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: saccharomyces cerevisiae. Expression_system_taxid: 4932

Resolution:

2.15Å R-factor: 0.212 R-free: 0.261

Authors:

C.I.Webster,M.Burrell,L.Olsson,S.B.Fowler,S.Digby,A.Sandercock, A.Snijder,J.Tebbe,U.Haupts,J.Grudzinska,L.Jermutus,C.Andersson

Key ref:

C.I.Webster et al. (2014). Engineering neprilysin activity and specificity to create a novel therapeutic for Alzheimer's disease. Plos One, 9, e104001. PubMed id: 25089527 DOI: 10.1371/journal.pone.0104001

Date:

13-Mar-14 Release date: 13-Aug-14

Protein chain

P08473  (NEP_HUMAN) -  Neprilysin from Homo sapiens

Seq: 750 a.a.

Struc: 697 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.24.11 - neprilysin.
• Reaction: Preferential cleavage at the amino group of hydrophobic residues in insulin, casein, hemoglobin, and a number of other proteins and polypeptides.
• Cofactor: Zn(2+)

DOI no: 10.1371/journal.pone.0104001

Plos One 9:e104001 (2014)

PubMed id: 25089527

Engineering neprilysin activity and specificity to create a novel therapeutic for Alzheimer's disease.

C.I.Webster, M.Burrell, L.L.Olsson, S.B.Fowler, S.Digby, A.Sandercock, A.Snijder, J.Tebbe, U.Haupts, J.Grudzinska, L.Jermutus, C.Andersson.

ABSTRACT

Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer's disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer's disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1-40, 1-42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1-40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer's disease.