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.

Keywords: Dehydration; Dehydroalanine; Dehydrobutyrine; Lantibiotic; Leader peptide; NisB dehydratase; NisC cyclase; Nisin.

Graphical abstract

Fig. 1

Schematic representation of the nisin biosynthesis and the nisin leader regions that were substituted with alanines (Table 2). A precursor nisin consists of the leader peptide part and a modifiable core peptide part. The leader peptide directs the precursor nisin to the nisin modification enzymes NisB and NisC and the transporter NisT. (A) The specific Ser/Thr residues in the core peptide part (highlighted in pink) are dehydrated to dehydroalanines (Dha) and dehydrobutyrines (Dhb), resulting in a dehydrated precursor nisin. (B) The dehydrated precursor nisin is a substrate for a cyclase NisC that catalyzes thioether bridges between the dehydrated amino acids and the downstream cysteines, resulting in a modified precursor nisin. (C) The modified precursor nisin is secreted outside the cell by the transporter NisT and the leader peptide is cleaved off by an extracellular protease NisP, liberating mature nisin. The figure demonstrates and is in accordance with recent recommendations for a universal nomenclature for ribosomally synthetized and posttranslationally modified peptide natural products [32]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Co-purification assay of NisB and NisC with precursor nisin and the precursor nisin leader mutants. (A) SDS–PAGE analysis. NisB is about 117.5 kDa. (B) Western blot with Anti-NisB antibodies. (C) Western blot with anti-NisC antibodies. The upper band corresponds to NisC bound to NisA [9]. Lane 1: Strain expressing wild-type enzymes NisBTC and a NisA-H₆ STKD(-22-19)AAAA. Lane 2: strain expressing wild-type enzymes NisBTC and a NisA-H₆ FNLD(-18-15)AAAA. Lane 3: strain expressing wild-type enzymes NisBTC and a NisA-H₆ LVSV(-14-11)AAAA. Lane 4: strain expressing wild-type enzymes NisBTC and a NisA-H₆ SKKD(-10-7)AAAA. Lane 5: strain expressing wild-type enzymes NisBTC and a NisA-H₆ SGAS(-6-3)AAAA. Lane 6: strain expressing wild-type enzymes NisBTC and a NisA-H₆ PR(-2-1)AA. Lane 7: strain expressing wild-type enzyme NisBTC and a NisA-H₆.