Requirements of the engineered leader peptide of nisin for inducing modification, export, and cleavage.

Abstract

Nisin A is a pentacyclic peptide antibiotic produced by Lactococcus lactis. The leader peptide of prenisin keeps nisin inactive and has a role in inducing NisB- and NisC-catalyzed modifications of the propeptide and NisT-mediated export. The highly specific NisP cleaves off the leader peptide from fully modified and exported prenisin. We present here a detailed mutagenesis analysis of the nisin leader peptide. For alternative cleavage, we successfully introduced a putative NisP autocleavage site and sites for thrombin, enterokinase, Glu-C, and factor Xa in the C-terminal part of the leader peptide. Replacing residue F-18 with Trp or Thr strongly reduced production. On the other hand, D-19A, F-18H, F-18M, L-16D, L-16K, and L-16A enhanced production. Substitutions within and outside the FNLD box enhanced or reduced the transport efficiency. None of the above substitutions nor even an internal 6His tag from positions -13 to -8 had any effect on the capacity of the leader peptide to induce NisB and NisC modifications. Therefore, these data demonstrate a large mutational freedom. However, simultaneous replacement of the FNLD amino acids by four alanines strongly reduced export and even led to a complete loss of the capacity to induce modifications. Reducing the leader peptide to MSTKDFNLDLR led to 3- or 4-fold dehydration. Taken together, the FNLD box is crucial for inducing posttranslational modifications.

• Authors: Annechien Plat, Leon D Kluskens, Anneke Kuipers, Rick Rink, Gert N Moll
• Journal: Applied and environmental microbiology (Appl Environ Microbiol) Vol. 77 Issue 2 Pg. 604-11 (Jan 2011) ISSN: 1098-5336 [Electronic] United States
• PMID: 21097596 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Anti-Bacterial Agents
• Protein Sorting Signals
• Nisin

Topics

• Amino Acid Sequence
• Amino Acid Substitution
• Anti-Bacterial Agents (metabolism, pharmacology)
• DNA Mutational Analysis
• Lactococcus lactis (genetics, metabolism)
• Microbial Sensitivity Tests
• Models, Molecular
• Molecular Sequence Data
• Nisin (genetics, metabolism, pharmacology)
• Protein Engineering
• Protein Processing, Post-Translational
• Protein Sorting Signals
• Protein Transport
• Recombination, Genetic