ACS Chemical Biology dehydrated AciA peptide (N-terminal 17 amino acids removed) was generated. AciA was ﬁrst oxidized to the corresponding disulﬁde, then treated with AciKC, and digested with Glu-C, which removed most of the leader peptide. This truncated peptide was then puriﬁed, reduced with TCEP, and incubated with AciKC for 1 h at RT. Subsequent ESI-MS−MS analysis showed that no labionin was formed in the truncated AciA peptide (Supplementary Figure S13). Thus, the leader peptide is essential for both dehydration and cyclization activity of class III lantipeptide synthetases. The formation of labionin is proposed to involve two tandem Michael addition reactions initiated by a C-terminal cysteine thiol.11 It is not yet known whether this process takes place in a stepwise manner with a lanthionine intermediate or whether both cross-links are made without any intermediates that are released from the enzyme. To further understand the cyclase activity of AciKC, a series of Ala mutations were introduced into the AciA peptide at positions that are precursors to labionin, and these mutant peptides were expressed hetero- logously in E. coli (Figure 6). The puriﬁed mutant peptides Figure 6. Fragmentation patterns of AciA mutant peptides modiﬁed by AciKC. All assays were performed in the presence of 5 mM NTP mixture, 10 mM MgCl2, and 0.1 mM TCEP. Modiﬁed peptides were digested by endoprotease Glu-C before further tandem MS analysis. were incubated with AciKC, ATP, and Mg2+, digested with endoprotease Glu-C, and subjected to ESI-MS−MS for ring structure analysis. As expected, when Ser11 was mutated to Ala, the formation of labionin A (generated from Dha8-Dha11- Cys15) was completely abolished (Figure 6, Supplementary Figure S14b). Similarly, incubation of AciA-S8A not only disrupted the formation of the carbon−carbon bond between residues 8 and 11 but also greatly impaired the formation of the thioether between Dha11 and Cys15 (Supplementary Figure S14a). This result suggests either a concerted mechanism for formation of both cross-links in a labionin, or that no active site acid is available to protonate the enolate intermediate to generate a lanthionine when the second Dha is not available. The formation of labionin B (Dha19-Dha22-Cys25) was not impacted by the loss of labionin A, as determined by tandem MS analysis. The eﬀect of mutation of Cys15 and Cys25 was remarkably diﬀerent (Figure 6, Supplementary Figure S14c,d). The formation of labionin A in the AciA-C15A peptide was disrupted as expected, whereas labionin B remained intact. Surprisingly, when Cys25 was mutated to Ala, it aﬀected eﬃcient formation of both labionins (Figure 6, Supplementary Figure S14d). These results suggest that formation of the C- terminal labionin B is important for the cyclization of labionin A but not vice versa. In turn, this suggests that cyclization may proceed in a C-to-N-terminal direction. This directionality would be opposite that observed for the class II lantibiotic Articles synthetases HalM2 and LctM,27 but it is similar to the recently reported directionality of the dehydration reaction of the class III lantipeptide synthetase LabKC,28 and that of a cyclo- dehydratase involved in the biosynthesis of another posttransla- tionally modiﬁed peptide containing thiazoles and oxazoles.29 Conclusions. In summary, by genome mining we discovered a novel class III lantipeptide catenulipeptin from Catenulispora acidiphila DSM 44928 to add to the growing number of class III lantipeptides. Like LabKC, the multifunc- tional enzyme AciKC catalyzes both dehydration and cyclization in vitro to form the labionin structures in catenulipeptin. AciKC shows promiscuity toward its NTP cosubstrate and can partially modify the AciA core peptide when the leader sequence is supplied in trans. The cyclase activity of AciKC resides in its C-terminal domain and does not require ATP or Zn ions. However, the leader of AciA peptide is required for cyclization. Furthermore, mutagenesis studies indicate that the formation of labionin A in catenulipeptin is dependent on the formation of the C-terminal labionin B. Thus far, catenulipeptin has not shown antimicrobial activity, but the compound can partially restore aerial hyphae growth of surfactin-treated S. coelicolor. It cannot restore hyphae formation in a bldA mutant. Whether its function in its producer strain is similar to that of SapB in S. coelicolor remains to be determined. Certainly, structurally very dissimilar compounds have been shown to be important in morpho- genesis in diﬀerent actinomycetes such as SapT30 and goadsporin.31,32 It has been suggested that these types of molecules play a strictly mechanical role that requires a surfactant activity, possibly by enforcing an amphipathic structure via the posttranslational modiﬁcations.33 ■ METHODS Materials. All oligonucleotides were purchased from Integrated DNA Technologies. Restriction endonucleases, DNA polymerases, and T4 DNA ligase were purchased from New England Biolabs or Invitrogen. Media components for bacterial cultures were purchased from Difco laboratories. Chemicals were purchased from Fisher Scienciﬁc or from Roche Biosciences. E. coli DH5α was used as host for cloning and plasmid propagation, and E. coli BL21 (DE3) was used as a host for expression of proteins and peptides. Cloning, Protein, and Peptide Expression. Detailed procedures are described in the Supporting Information. Primer sequences for peptide mutants are included in Supplementary Table S1. The protein sequence of AciKC is available in the National Center for Biotechnology Information (NCBI) database under accession number YP_003114944. Production of Catenulipeptin from C. acidiphila. Catenulispora acidiphila DSM 44928 was cultivated in trypticase soy broth liquid medium, pH 5.5−6.0, at 30 °C for 5−7 days. The supernatant of the culture was lyophilized to dryness, and the lantipeptide was extracted with acetonitrile. Enzyme Activity Assay. In a typical experiment, AciA substrate (30 μM) was incubated with AciKC (3 μM) in the presence of TCEP (0.1 mM), MgCl2 (10 mM), and NTP (5 mM) at RT in 50 mM Tris buﬀer (pH 8.0) for 4 h. The reaction was quenched by addition of TFA and analyzed by MALDI-TOF MS. Detection of Labionin by GC−MS. The protocol was adopted and modiﬁed from ref 18. Lyophilized AciKC-modiﬁed AciA (0.8 mg) was dissolved in 6 M hydrochloric acid (2 mL) and heated at 110 °C in a high-pressure, sealed vessel for 20 h. The reaction was cooled to RT, and the hydrochloric acid was removed under a gentle stream of nitrogen. The dry hydrolysate was then dissolved in 500 μL of 2 M ethanolic HCl, prepared from acetylchloride in ethanol (1:4, v:v). The sample was heated at 110 °C for 15 min, and reagents were again removed under a gentle stream of nitrogen. To the resulting sample 1533 dx.doi.org/10.1021/cb3002446 | ACS Chem. Biol. 2012, 7, 1529−1535