Evolution of lanthipeptide synthetases Qi Zhanga,YiYub, Juan E. Vélasqueza, and Wilfred A. van der Donka,b,c,1 aDepartment of Chemistry, bDepartment of Biochemistry, and cHoward Hughes Medical Institute, University of Illinois at Urbana–Champaign, Urbana, IL 61801 Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved September 24, 2012 (received for review June 19, 2012) Lanthionine-containing peptides (lanthipeptides) are a family of cyclase domain. Class III and class IV lanthipeptides are synthesized ribosomally synthesized and posttranslationally modified peptides by trifunctional enzymes LanKC (10, 13) and LanL (12), respectively. containing (methyl)lanthionine residues. Here we present a phylo- These enzymes contain an N-terminal lyase domain and a central genomic study of the four currently known classes of lanthipep- kinase domain but differ in their C termini. LanC and the C-terminal tide synthetases (LanB and LanC for class I, LanM for class II, LanKC domains of LanM and LanL contain a conserved zinc-binding motif for class III, and LanL for class IV). Although they possess very (Cys-Cys-His/Cys), whereas the C-terminal cyclase domain of LanKC similar cyclase domains, class II–IV synthetases have evolved inde- lacks these conserved residues (13) (Fig. 1B). pendently, and LanB and LanC enzymes appear to not always have Recent genome mining studies have revealed that lanthipeptide coevolved. LanM enzymes from various phyla that have three cys- biosynthetic genes are present in a wide range of bacteria (14–16). teines ligated to a zinc ion (as opposed to the more common Cys- The widespread occurrence may not be surprising, given the po- Cys-His ligand set) cluster together. Most importantly, the phylo- tential benefits of gene-encoded natural products with respect to genomic data suggest that for some scaffolds, the ring topology of facile evolution of new structures and biological function. Here we fi the nal lanthipeptides may be determined in part by the se- present a systematic analysis of the phylogenetic distribution of quence of the precursor peptides and not just by the biosynthetic lanthipeptide synthetases. We correlate the taxonomy of the bac- enzymes. This notion was supported by studies with two chimeric terial host and the structure of the final products with the enzyme peptides, suggesting that the nisin and prochlorosin biosynthetic phylogenies. Implications for biosynthetic engineering of the lan- enzymes can produce the correct ring topologies of epilancin 15X thipeptide family and for genome mining are discussed. and lacticin 481, respectively. These results highlight the potential of lanthipeptide synthetases for bioengineering and combinatorial Results and Discussion biosynthesis. Our study also demonstrates unexplored areas of Evolution of LanC enzymes. LanCs have about 400 amino acid res- sequence space that may be fruitful for genome mining. idues, and possess a double α–barrel-fold topology (17) and a molecular evolution | natural products | phylogeny | strictly conserved Cys-Cys-His triad near their C termini for binding posttranslational modification | lantibiotics of a zinc ion. In vitro reconstitution of the nisin cyclase activity of NisC and solution of its crystal structure have supported a zinc- dependent mechanism (17, 18). The zinc ion is believed to activate eptide antibiotics represent a large and diverse group of bio- the Cys thiols of the precursor peptide for nucleophilic attack on active natural products with a wide range of applications. Most P the dehydroamino acids. LanC or LanC-like enzymes are not only of these compounds are produced via two distinct biosynthetic found as stand-alone cyclases and as cyclase domains in LanM and paradigms. The nonribosomal peptide synthetases are responsible for the biosynthesis of many clinically important antibiotics (1, 2). LanL enzymes, their encoding genes are also present in eukaryotes, A different strategy involves posttranslational modifications of although the detailed functions of these LanC-like (LanCL) pro- linear ribosomally synthesized peptides (3, 4). This biosynthetic teins remain to be determined (19). strategy is also widely distributed and found in all three domains of The phylogeny of LanC from different bacterial lineages, in- life. Although the building blocks used by ribosomes are generally cluding the C-terminal domains of LanMs and LanLs, and the confined to the 20 proteinogenic amino acids, the structural di- LanCL proteins from human that can serve as the outgroup, was versity generated by posttranslational modifications is vast (5). constructed using both Bayesian Markov chain Monte Carlo Among the best-studied ribosomally synthesized and posttransla- (MCMC) (20) and maximum-likelihood inferences (21). To obviate tionally modified peptides are the lanthipeptides, a class of com- codon bias, the trees were constructed based on amino acid pounds distinguished by the presence of sulfur-to-β–carbon thioether sequences instead of nucleic acid sequences. The overall Bayesian cross-links named lanthionines and methyllanthionines (Fig. 1) (6–9). MCMC tree shown in Fig. 2 A and B is almost exactly the same with Many lanthipeptides, such as the commercially used food pre- that prepared by the maximum-likelihood method (SI Appendix, servative nisin, have potent antimicrobial activity and are termed Fig. S1), strongly supporting the reliability of both trees. lantibiotics. Maturation of lanthipeptides involves posttranslational The C-terminal domains of LanM and LanL fall into two distinct modifications of a C-terminal core region of a precursor peptide and clades. These two clades group into a larger clade and are sepa- subsequent proteolytic removal of an N-terminal leader sequence rated from a sister LanC clade and the eukaryotic LanCL clade that is not modified (3). Their thioether bridges are installed by the (Fig. 2A and SI Appendix, Fig. S1), suggesting that LanM and LanL initial dehydration of Ser and Thr residues, followed by stereo- evolved independently from LanC. If LanM and LanL originate selective intramolecular Michael-type addition of Cys thiols to the from hybridization of an ancestral LanC with a dehydratase or newly formed dehydroamino acids (Fig. 1A). In some cases, this re- kinase, this event likely occurred only once. Gene recombination action is coupled with a second Michael-type addition of the resulting CHEMISTRY enolate to a second dehydroalanine to produce a labionin structure (10). Genetic and biochemical studies have revealed four distinct Author contributions: Q.Z., Y.Y., J.E.V., and W.A.v.d.D. designed research; Q.Z., Y.Y., and classes of lanthipeptides according to their biosynthetic machinery (7, J.E.V. performed research; Q.Z., Y.Y., J.E.V., and W.A.v.d.D. analyzed data; and Q.Z. and 11, 12) (Fig. 1B). Class I lanthipeptides are synthesized by two dif- W.A.v.d.D. wrote the paper. ferent enzymes, a dehydratase LanB, and a cyclase LanC (Lan is The authors declare no conflict of interest. a generic designation for lanthipeptide biosynthetic proteins). For This article is a PNAS Direct Submission. class II lanthipeptides, the reactions are carried out by a single lan- 1To whom correspondence should be addressed. E-mail: [email protected]. thipeptide synthetase, LanM, containing an N-terminal dehydratase This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. BIOCHEMISTRY domain that bears no homology to LanB, and a C-terminal LanC-like 1073/pnas.1210393109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1210393109 PNAS | November 6, 2012 | vol. 109 | no. 45 | 18361–18366 Downloaded by guest on September 30, 2021 A few lanthipeptide biosynthetic gene clusters contain genes encoding a C-terminally truncated LanB and a stand-alone SpaB_C-like protein, suggesting that the two domains of LanB might be able to act both in cis and in trans. Similarly, most of the thiopeptide biosynthetic gene clusters encode two proteins for dehydration: a putative dehydratase that shares low se- quence similarity with LanBs and a SpaB_C-like protein (27). In this study, only full-length LanB sequences were used. The phylogenetic trees of LanB enzymes from the same gene cluster as the LanC proteins in Fig. 2B were constructed by both Bayesian MCMC and maximum-likelihood methods, and the resulting two trees are almost identical (SI Appendix, Figs. S4 and S5). Interestingly, the topology of the LanB tree is distinct from that of LanC. The enzymes from bacteroidetes and proteobac- teria are possibly derived from firmicutes because they are deeply nested within a group that mainly consists of LanBs from firmi- cutes (SI Appendix, Fig. S4). This finding is in distinct contrast to Fig. 1. Biosynthesis of lanthipeptides, showing the mechanism of (methyl) the LanC tree in which the enzymes of group 1 are distantly re- lanthionine formation (A), and the four classes of synthetases (B). Xn rep- lated to those of group 3 (Fig. 2B). It appears therefore that for resents a peptide linker. The conserved zinc-binding motifs are highlighted by the purple lines in the cyclase domains. SpaB_C is an in silico defined these clusters, LanBs have evolved independently from LanCs, or domain currently found as a stand-alone protein for thiopeptide bio- that the LanBs or LanCs may have been recruited from other synthesis and as the C-terminal domain of LanB enzymes. The N-terminal organisms and have formed a functional pair. In general, how- domain of LanB enzymes is made up of two subdomains according to the ever, the trees show that LanB and LanC enzymes from the same Conserved Domain Database (26). organism fall into similar clades (Fig. 2B and SI Appendix,Fig. S4), suggesting that they have coevolved. Genome mining for new class I lanthipeptides might be facilitated between the C-terminal domains of LanM and LanL is not sup- by phylogenetic categorization of LanC and LanB enzymes. For ported by our analysis of the currently available sequences. example, we note that Streptococcus pasteurianus contains genes for LanCs from different bacterial phyla group into three groups LanB and LanC that are phylogenetically closely related to NisB with strong statistical support (Fig.