Investigation of Substrate Recognition and Biosynthesis in Class IV Lanthipeptide Systems

Abstract

Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are subdivided into four classes. The first two classes have been heavily studied, but less is known about classes III and IV. The lanthipeptide synthetases of classes III and IV share a similar organization of protein domains: A lyase domain at the N-terminus, a central kinase domain, and a C-terminal cyclase domain. Here, we provide deeper insight into class IV enzymes (LanLs). A series of putative producer strains was screened to identify production conditions of four new venezuelin-like lanthipeptides, and an Escherichia coli based heterologous production system was established for a fifth. The latter not only allowed production of fully modified core peptide but was also employed as the basis for mutational analysis of the precursor peptide to identify regions important for enzyme recognition. These experiments were complemented by in vitro binding studies aimed at identifying the region of the leader peptide recognized by the LanL enzymes as well as determining which domain of the enzyme is recognizing the substrate peptide. Combined, these studies revealed that the kinase domain is mediating the interaction with the precursor peptide and that a putatively α-helical stretch of residues at the center to N-terminal region of the leader peptide is important for enzyme recognition. In addition, a combination of in vitro assays and tandem mass spectrometry was used to elucidate the order of dehydration events in these systems.

Figure 1

Overview of (a) different lanthipeptide classes and (b) mechanisms of lanthipeptide biosynthesis.

Figure 2

(a) General organization of class IV lanthipeptide biosynthetic gene clusters (b) Alignment of the venezuelin (VenA) and streptocollin (StcA) precursor peptides with the precursor peptides of the clusters investigated in this study. Residues undergoing dehydration are highlighted in blue, cysteines involved in ring formation in orange. According to standard RiPP nomenclature, the first amino acid of the core peptide is designated as residue 1, while the last amino acid of the leader peptide is defined as residue −1. (c) Primary structures of venezuelin and streptocollin. Abu = aminobutyric acid.

Figure 3

(a) Schematic representation of the heterologous E. coli production system for the B2293 lanthipeptide globisporin. (b) Amino acid sequence of the last 29 residues of the SgbA precursor peptide showing the cyclization pattern known from venezuelin and streptocollin. Positions where b and y fragments were detected by tandem MS (including mass shifts due to water losses) are highlighted. (c) Tandem MS of the −4 H₂O species of the extracted 29 amino acid (aa) peptide. The lack of detectable fragments in the region of Thr2-Cys21 (and overall low intensity of fragment ions) suggests formation of a methyllanthionine ring between these residues. For calculated and observed m/z values see Supporting Information Table S3. The tandem MS data only provides evidence of an overlapping ring topology that spans Dhb2 to Cys21. The topology the rings shown in (b) was predicted based on the high similarity of the globisporin system with the sequences producing venezuelin and streptocollin (Figure 2).

Figure 4

(a) Schematic representation of the heterologous E. coli production system for His₆-MBP-SgbA co-expressed with SgbL. (b) Overview of all variants of His₆-MBP-SgbA tested in the co-expression construct (see also Supporting Information Figure S6).

Figure 5

(a) Schematic showing the principle of the fluorescence polarization (FP) binding studies. The representative graph was recorded at 100 nM FITC-SgbA(leader) and varying concentrations of His₆-SgbL. (b) Overview of the results of the FP binding studies with the different N-terminally FITC-labeled leader peptide truncants. Predicted α-helical regions in the precursor peptide are shown in red. The corresponding graphs are depicted in Supporting Information Figures S7 and S8 (c) Overview of the results of the FP competition assays using 100 nM FITC-SgbA(leader), 240 nM His₆-SgbL and varying concentrations of the respective competitor peptide.

Figure 6

Tandem MS analysis of the different trypsin-treated, dehydrated species of His₆-SgbA. If no mass change was observed for a specific b or y fragment after a single dehydration event, they are highlighted in blue or red, respectively. If a shift of −18 Da is observed for a specific b or y fragment, compared to the preceding +1 H₂O species, they are colored in green or orange. It is apparent that the His₆-SgbL(lyase-kinase 1-548) catalyzed dehydration starts at Thr2, proceeds to Thr10, continues to Thr14 and ends with modification of Ser20. The absence of some of the last b and first y fragments for the four-fold dehydrated peptide suggests that during the overnight incubation to obtain this species non-enzymatic cyclization of Cys16 and Dha20 occurred, which is in line with the NEM labeling results (Supporting Information Figure S11). For calculated and observed m/z values see Supporting Information Table S5.

Figure 7

FP assays using 100 nM FITC-SgbA(leader) and varying concentrations of the respective SgbL domains.