title: 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.affiliation: BiOMaDe Technology Foundation, Nijenborgh 4, 9747 AG Groningen, Netherlands.
authors.forename: Annechien
authors.initials: A
authors.lastname: Plat
authors.forename: Leon D
authors.initials: LD
authors.lastname: Kluskens
authors.forename: Anneke
authors.initials: A
authors.lastname: Kuipers
authors.forename: Rick
authors.initials: R
authors.lastname: Rink
authors.forename: Gert N
authors.initials: GN
authors.lastname: Moll
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D004252:DNA Mutational Analysis; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009561:Nisin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D021381:Protein Transport; D011995:Recombination, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D021382:Protein Sorting Signals; D009561:Nisin