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.

FIG. 1.

Schedule of nisin biosynthesis involving NisB-mediated dehydration of Ser and Thr in the propeptide, NisC-catalyzed cyclization, NisT-mediated export, and NisP-mediated removal of the leader peptide.

FIG. 2.

Leader peptides containing the F(N/D)LD box were aligned by use of the ClustalW2 program (European Bioinformatics Institute, Cambridge, United Kingdom). Streptin was aligned arbitrarily. Colons, conserved substitutions; periods, semiconserved substitutions; asterisk, identical residues. The numbers in parentheses on the right indicate the reference.

FIG. 3.

Production analysis of FNLD mutants. (A) Supernatants of L. lactis containing pIL3BTC producing wild-type prenisin and FNLD box mutants. Peptide was analyzed by PageBlue-stained gel electrophoresis. Lanes: AAAA, quadruple mutation F-18A/N-17A/L-16A/D-15A; AALD, double mutation F-18A/N-17A; FNAA, double mutation L-16A/D-15A; WT, wild type prenisin A. (B) Prenisin mutants detected by anti-leader peptide antibody in cell extracts from L. lactis containing pIL3BTC. WT, wild type prenisin A; FNAA, double mutation L-16A/D-15A. (C) Supernatants of L. lactis containing pIL3BTC producing wild-type prenisin (WT) and prenisin containing a point mutation. Peptide was analyzed by PageBlue-stained gel electrophoresis.

FIG. 4.

Export of prenisin with quadruple mutations (F-18A/N-17A/L-16A/D-15A) without dehydration by L. lactis containing pIL3BTC. Peptide export was detected by MALDI-TOF mass spectrometric analysis of the culture supernatant, after incubation with 1 mg/ml TCEP to prevent cysteinylation. The [M + H⁺] peak of 5,625.11 corresponds to unmodified prenisin without an initial methionine (5,627.58 Da). The difference of −2 Da could be a result of disulfide bond formation.

FIG. 5.

Effect of mutations on export efficiency. Production levels are relative to the level of wild-type prenisin A production. Small characters indicate lower levels of production, medium-size characters indicate levels of production similar to the level of production of wild-type nisin, and large characters indicate higher levels of production.

FIG. 6.

Export of prenisin with shortened leader peptides containing the FNLD box by L. lactis containing pIL3BTC. Peptide export was detected by MALDI-TOF mass spectrometric analysis of the culture supernatant, after incubation with 1 mg/ml TCEP to prevent cysteinylation. (A) MFNLDLR-nisin. The [M + H⁺] peaks of 4,390.64 and 4,257.31 correspond to unmodified prenisin with an initial methionine (4,389.28 Da) and without an initial methionine (4,258.08 Da), respectively. Peak 4,417.24 corresponds to formylated (—CHO) MFNLDLR-nisin (4,417.29 Da). (B) MSTKDFNLDLR-nisin. The [M + H⁺] peaks of 4,616.98 and 4,635.62 correspond to prenisin without an initial methionine 4-fold dehydrated (4,617.47 Da) and 3-fold dehydrated (4,635.49 Da), respectively. The peak of 4,820.50 corresponds to undehydrated prenisin with an initial methionine (4,820.72 Da). (C) MSTKDFNLDLR-ITSISLCTPGCKTG is dehydrated once. Peptide masses correspond to prenisin with an initial methionine dehydrated once (2,685.14 Da) and not dehydrated (2,703.16 Da). Subsequent peaks are again once and not dehydrated prenisin but contain a formylated initial methionine, resulting in an additional mass of 28 Da. The [M + H⁺] peak of 2,747.34 could represent either an oxidation (+16 Da) or another dehydration (+18 Da).