Lantibiotics are (methyl)lanthionine-containing bacterial peptides. (Methyl)lanthionines are posttranslationally introduced into the prepropeptides by biosynthetic enzymes that dehydrate serines and threonines and couple these dehydrated residues to cysteine residues. Thirty seven lantibiotic primary structures have been proposed to date, but little is known about the substrate specificity of the lantibiotic modifying enzymes. To define rules for the rational design of modified peptides, we compared all known lantibiotic structures by in silico analysis. Although no strict sequence motifs can be defined that govern the modification, statistical analysis demonstrates that dehydratable serines and threonines are more often flanked by hydrophobic than by hydrophilic amino acids. Serine residues escape dehydration more often than threonines. With these rules, novel hexapeptides were designed that either were predicted to become modified or will escape modification. The hexapeptides were fused to the nisin leader and expressed in a Lactococcus lactis strain containing the nisin modifying and export enzymes. The excreted peptides were analyzed by mass spectrometry. All designed fusion peptides were produced, and the presence or absence of modifications was found to be in full agreement with the predictions based on the statistical analysis. These findings demonstrate the feasibility of the rational design of a wide range of novel peptides with dehydrated amino acid residues.

Lantibiotics are lanthionine-containing peptide antibiotics. Nisin, encoded by nisA, is a pentacyclic lantibiotic produced by some Lactococcus lactis strains. Its thioether rings are posttranslationally introduced by a membrane-bound enzyme complex. This complex is composed of three enzymes: NisB, which dehydrates serines and threonines; NisC, which couples these dehydrated residues to cysteines, thus forming thioether rings; and the transporter NisT. We followed the activity of various combinations of the nisin enzymes by measuring export of secreted peptides using antibodies against the leader peptide and mass spectroscopy for detection. L. lactis expressing the nisABTC genes efficiently produced fully posttranslationally modified prenisin. Strikingly, L. lactis expressing the nisBT genes could produce dehydrated prenisin without thioether rings and a dehydrated form of a non-lantibiotic peptide. In the absence of the biosynthetic NisBC enzymes, the NisT transporter was capable of excreting unmodified prenisin and fusions of the leader peptide with nonlantibiotic peptides. Our data show that NisT specifies a broad spectrum (poly)peptide transporter that can function either in conjunction with or independently from the biosynthetic genes. NisT secretes both unmodified and partially or fully posttranslationally modified forms of prenisin and non-lantibiotic peptides. These results open the way for efficient production of a wide range of peptides with increased stability or novel bioactivities.

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.

Nisin A is a pentacyclic antibiotic peptide produced by various Lactococcus lactis strains. Nisin displays four different activities: (i) it autoinduces its own synthesis; (ii) it inhibits the growth of target bacteria by membrane pore formation; (iii) it inhibits bacterial growth by interfering with cell wall synthesis; and, in addition, (iv) it inhibits the outgrowth of spores. Here we investigate the structural requirements and relevance of the N-terminal thioether rings of nisin by randomization of the ring A and B positions. The data demonstrate that: (i) mutation of ring A results in variants with enhanced activity and a modulated spectrum of target cells; (ii) for the cell growth-inhibiting activity of nisin, ring A is rather promiscuous with respect to its amino acid composition, whereas the bulky amino acid residues in ring B abolish antimicrobial activity; (iii) C-terminally truncated nisin A mutants lacking rings D and E retain significant antimicrobial activity but are unable to permeabilize the target membrane; (iv) the dehydroalanine in ring A is not essential for the inhibition of the outgrowth of Bacillus cells; (v) some ring A mutants have significant antimicrobial activities but have decreased autoinducing activities; (vi) the opening of ring B eliminates antimicrobial activity while retaining autoinducing activity; and (vii) some ring A mutants escape the nisin immune system(s) and are toxic to the nisinproducing strain NZ9700. These data demonstrate that the various activities of nisin can be engineered independently and provide a basis for the design and synthesis of tailor-made analogs with desired activities.Lantibiotics are (methyl)lanthionine-containing antibiotics (1, 9) produced by some gram-positive bacteria. The lanthionines are posttranslationally formed by enzyme-mediated dehydration of serine and threonine residues followed by enzymecatalyzed intramolecular coupling to cysteines to form a thioether bridge. The lantibiotic nisin contains one lanthionine and four methyllanthionines (Fig. 1). Nisin autoregulates its own synthesis (19) by binding to the transmembrane protein NisK, which phosphorylates the intracellular response regulator NisR. Phosphorylated NisR activates the nis promoter.Nisin predominantly acts against gram-positive bacteria. This occurs at nanomolar concentrations as a consequence of the interaction of nisin with the docking molecule lipid II (4, 6). Rings A and B physically interact with lipid II, and this results in membrane permeabilization by hybrid pores of nisin and lipid II (5) and inhibition of cell wall synthesis via lipid II abduction (11). Nisin-producing bacteria are protected against nisin by two self-protection mechanisms: a lipoprotein, NisI, which likely binds and inactivates nisin, and an export system, NisEFG, which presumably extrudes nisin from the cell (20). Another antibiotic activity of nisin is the inhibition of the outgrowth of spores. Nisin's dehydroalanine in position 5 has been reported to be involved in this inhibitory activity (29). R...

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.