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Identification of distinct nisin leader peptide regions that determine interactions with the modification enzymes NisB and NisC.

ABSTRACT: Nisin is the most prominent and applied bacteriocin that serves as a model for class I lantibiotics. The nisin leader peptide importantly determines interactions between precursor nisin and its modification enzymes NisB and NisC that mature nisin posttranslationally. NisB dehydrates serines and threonines, while NisC catalyzes the subsequent coupling of the formed dehydroamino acids to form lanthionines. Currently, little is known about how the nisin leader interacts with NisB and even less is known about its interactions with NisC. To investigate the nisin leader peptide requirements for functional interaction with the modification enzymes NisB and NisC, we systematically replaced six regions, of 2-4 amino acids each, with all-alanine regions. By performing NisB and NisC co-purification studies with these mutant leader peptides, we demonstrate that the nisin leader regions STKD(-22-19), FNLD(-18-15) and PR(-2-1) importantly contribute to the interactions of precursor nisin with both NisB and NisC, whereas the nisin leader region LVSV(-14-11) additionally contributes to the interaction of precursor nisin with NisC.

SUBMITTER: Khusainov R

PROVIDER: S-EPMC3678300 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification of distinct nisin leader peptide regions that determine interactions with the modification enzymes NisB and NisC.

Khusainov Rustem R Moll Gert N GN Kuipers Oscar P OP

FEBS open bio 20130530

Nisin is the most prominent and applied bacteriocin that serves as a model for class I lantibiotics. The nisin leader peptide importantly determines interactions between precursor nisin and its modification enzymes NisB and NisC that mature nisin posttranslationally. NisB dehydrates serines and threonines, while NisC catalyzes the subsequent coupling of the formed dehydroamino acids to form lanthionines. Currently, little is known about how the nisin leader interacts with NisB and even less is k ...[more]

PMID: 23772400

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Project description:Nisin produced by Lactococcus lactis 6F3 is used as a food preservative and is the most important member of a group of peptide-antibiotics containing lanthionine bridges (lantibiotics) (N. Schnell, K.-D. Entian, U. Schneider, F. Götz, H. Zähner, R. Kellner, and G. Jung, Nature [London] 333:276-278, 1988). Nisin is ribosomally synthesized, and its structural gene, nisA, encodes a prepeptide that is posttranslationally modified, revealing the active lantibiotic (C. Kaletta and K.-D. Entian, J. Bacteriol. 171:1597-1601, 1989). Adjacent to nisA, the additional genes nisB, nisT, and nisC were identified. Over their entire sequences, these genes were homologous to genes recently identified as important for the biosynthesis of lantibiotics, that is, subtilin from Bacillus subtilis ATCC 6633 and epidermin from Staphylococcus epidermidis Tü 3298. Genes nisB, nisT, and nisC corresponded to open reading frames of 993, 600, and 418 amino acid residues, respectively. The nisT open reading frame is homologous to proteins of the HlyB (hemolysin B protein of Escherichia coli) subfamily. Proteins of this subfamily are responsible for the secretion of a variety of compounds, including large polypeptides, polysaccharides, and anti-drug tumors, indicating that NisT may be involved in nisin transport. Northern (RNA) blot analysis revealed a 0.3-kb transcript for the nisA structural gene, and the transcriptional start point of the nisA gene was determined by primer extension. Additionally, a mRNA of at least 3 kb was identified by using a hybridization probe specific to nisB. Antibodies were raised against the NisB protein, and Western blot (immunoblot) analysis revealed a molecular weight of about 115 kDa, which is in accordance with the theoretical protein size of 117.5 kDa as calculated from the nisB open reading frame. Several amphipathic transmembrane alpha-helices indicated that NisB is associated with the membrane. This was confirmed by preparing L. lactis vesicles. The NisB protein was tightly associated with the vesicle fraction and was released by sodium dodecyl sulfate treatment only. These results suggest that NisB is membrane associated and that nisin biosynthesis occurs at the cell membrane.

Dataset Information

Identification of distinct nisin leader peptide regions that determine interactions with the modification enzymes NisB and NisC.

Publications

Identification of distinct nisin leader peptide regions that determine interactions with the modification enzymes NisB and NisC.

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