Directionality and coordination of dehydration and ring formation during biosynthesis of the lantibiotic nisin

Abstract

The lantibiotic nisin is a potent antimicrobial substance, which contains unusual lanthionine rings and dehydrated amino acid residues and is produced by Lactococcus lactis. Recently, the nisin biosynthetic machinery has been applied to introduce lanthionine rings in peptides other than nisin with potential therapeutic use. Due to difficulties in the isolation of the proposed synthetase complex (NisBTC), mechanistic information concerning the enzymatic biosynthesis of nisin is scarce. Here, we present the molecular characterization of a number of nisin mutants that affect ring formation. We have investigated in a systematic manner how these mutations influence dehydration events, which are performed enzymatically by the dehydratase NisB. Specific mutations that hampered ring formation allowed for the dehydration of serine residues that directly follow the rings and are normally unmodified. The combined information leads to the conclusion that 1) nisin biosynthesis is an organized directional process that starts at the N terminus of the molecule and continues toward the C terminus, and 2) NisB and NisC are alternating enzymes, whose activities follow one after another in a repetitive way. Thus, the dehydration and cyclization processes are not separated in time and space. On the basis of these results and previous knowledge, a working model for the sequence of events in the maturation of nisin is proposed.

FIGURE 1.

Primary structure of prenisin and generated mutants. Dehydrated residues are shaded gray; serine 33 sometimes escapes dehydration and is shaded light gray. Serine at position 29 is never dehydrated in wild type prenisin. The impact of mutations on the dehydration pattern of new prenisin species is schematically depicted. Mutated residues are indicated by filled red circles. Newly formed dehydrated residues are pointed to by a black arrow. Letters A–E correspond to the five consecutive lanthionine rings in nisin.

FIGURE 2.

Examples of mass spectra of prenisin and prenisin mutants produced by L. lactis NZ9000 harboring pIL3BTC plasmid required for expression of nisin modification machinery and various pNZnisA-E3 plasmids encoding for prenisin and its mutants. Visualized are mass peaks corresponding to species of prenisin or its mutants without initial methionine (L. lactis usually removes initial methionine from synthesized peptides) with different numbers of dehydrated residues indicated by roman numerals. Other major peaks represent prenisin species containing the initial methionine with different degrees of dehydration (the dehydration pattern of peptides with and without initial methionine is identical), which were not labeled due to clarity.

FIGURE 3.

Mass spectra of prenisin mutants produced by L. lactis NZ9000 harboring pIL3BTC plasmid required for expression of nisin modification machinery and various pNZnisA-E3 plasmids encoding for prenisin mutants. Visualized are mass peaks corresponding to species of prenisin or its mutants without initial methionine (L. lactis usually removes initial methionine from synthesized peptides) with different numbers of dehydrated residues indicated by roman numerals. Other major peaks represent prenisin specious containing initial methionine with different degrees of dehydration (the dehydration pattern of peptides with and without initial methionine is identical), which were not labeled due to clarity.

FIGURE 4.

Mass spectra of prenisin produced by L. lactis NZ9000 harboring pIL3BTC^H331A plasmid required for expression of nisin modification machinery, with functionally truncated NisC and pNZnisA-E3 plasmids encoding for prenisin. A, two major mass peaks corresponding to prenisin (without initial methionine) dehydrated 8- and 9-fold. B, two major mass peaks corresponding to nisin (without initial methionine), dehydrated 8- and 9-fold.

FIGURE 5.

Mass spectra of prenisin mutants produced by L. lactis NZ9000 harboring the pIL3BTC^H331A plasmid required for expression of the nisin modification machinery, with functionally truncated NisC and derivatives of pNZnisA-E3 plasmids encoding for prenisin mutants. Shown are major mass peaks (without initial methionine) obtained for various mutants. Panel A shows mass peaks for mutant K12S, and panel B shows mass peaks for mutant N20S.

FIGURE 6.

Working model of nisin biosynthesis. A, prenisin is synthesized ribosomally. B, prenisin leader is recognized and bound by lanthionine synthetase NisBTC. C–E, nisin is pulled, possibly by the transporter NisT, through the active site of NisB and NisC. NisC and NisB alternate in order to install lanthionine rings. First NisB (as indicated schematically by an asterisk) dehydrates available serines and threonines (C), and then NisC (indicated by an asterisk) cyclizes the dehydrated residue with an appropriate cysteine (D). E, subsequently, prenisin is pulled, possibly by NisT, and new rings can be installed consecutively. F, next, modified prenisin is transported across the lipid bilayer by NisT. In wild type, the precursor is then cleaved by NisP, yielding the active antimicrobial peptide.