Heterologous expression and characterization of recombinant Lactococcus lactis neutral endopeptidase (neprilysin)

Wei Lian, Donghai Wu, Wil N. Konings, Igor Mierau, Louis B. Hersh

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

A neutral endopeptidase (NEP) from Lactococcus lactis has recently been cloned and shown to contain high sequence homology with the human neutral endopeptidase, endopeptidase 24.11 (I. Mierau et al., J. Bacteriol. 175, 2087-2096, 1993). The gene for the neutral endopeptidase from L. lactis was cloned into the pQE expression vector, resulting in the fusion of a hexahistidine at the N-terminus. The recombinant enzyme was expressed to high levels in Escherichia coli (~10 mg/liter of culture) and purified to homogeneity in a two-step procedure. A number of peptides were studied as substrates for the enzyme. The enzyme cleaves the following peptides at the Gly3-Phe4 bond: enkephalins, dynorphins A-6, A-8, A-9, A-10, A-13, and A- 17, and α-neo-endorphin. In addition the enzyme hydrolyzes bradykinin, substance P, β-endorphin, ACTH, and VIP. Although the cleavage patterns observed are similar to that seen with mammalian neutral endopeptidase, the lactococcal enzyme more efficiently cleaves larger peptide substrates. As observed with the mammalian neutral endopeptidase, the lactococcal enzyme exhibits higher k(cat)/K(m) values for the enkephalins than for their corresponding amides, indicating the functionality of an active-site arginine. Inactivation of the lactococcal endopeptidase by diethyl pyrocarbonate and protection afforded by the substrate dynorphin A-6 indicate the functionality of a positionally conserved active-site histidine. This was confirmed by demonstrating that conversion of this histidine, histidine 587, to glutamine generated inactive enzyme. Similarly, conversion of the putative zinc ligand glutamate 535 to glutamine led to inactive enzyme. These studies indicate a conservation of critical catalytic residues between the two enzymes and suggest that the lactococcal endopeptidase is a better model than thermolysin for the mammalian enzyme.

Original languageEnglish
Pages (from-to)121-126
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume333
Issue number1
DOIs
StatePublished - Sep 1 1996

Bibliographical note

Funding Information:
This work was supported in part by a grant from the National Institute on Drug Abuse, DA 02243.

Keywords

  • active site
  • endopeptidase
  • opioid peptides
  • substrate specificity

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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