ACS Chemical Biology mass, as determined by MALDI-TOF MS, suggesting that the two Cys residues had been converted to nonreactive thioethers (Supplementary Figure S3). This conclusion was further supported by tandem mass spectrometry. AciKC-modiﬁed AciA was digested by endoprotease Glu-C to partially remove the leader peptide, and the product was subjected to ESI-MS− MS analysis. A similar fragmentation pattern was observed as that of the mature catenulipeptin from culture extract (Supplementary Figure S4a). These results show that AciKC is a multifunctional enzyme that catalyzes both dehydration and cyclization reactions. Structure and Bioactivity of Catenulipeptin. In order to conﬁrm the formation of labionins during the in vitro assay, AciKC-modiﬁed AciA peptide was hydrolyzed in acid, and the resulting amino acids were derivatized to the corresponding N- triﬂuoroacetamido ethyl esters by a previously reported procedure.18 The resulting material was analyzed by gas chromatography (GC) coupled to positive chemical ionization (PCI) MS, resulting in two peaks with retention times of 11.24 and 11.32 min (Supplementary Figure S5a). The molecular ions associated with these peaks are consistent with those reported previously for derivatized labionin.18 Furthermore, the fragment ions match those previously reported (Supplementary Figure S5b).18 The observation of two peaks is the result of epimerization of labionin during hydrolysis as previously reported.18 Unlike a very recent report on new class III lantipeptides from actinomycetes that contained both labionin and lanthionine,17 catenulipeptin produced in vitro did not contain lanthionine derivatives as determined by GC−MS.19,20 Combined with the ESI-MS−MS data, we conclude that like LabKC but unlike RamC, AciKC installs labionins in its substrate peptide. Labionin B in catenulipeptin is somewhat unusual in that it has only two residues between Cys25 and Ser22 rather than the three or ﬁve residues found thus far in other class III lantipeptides that contain labionins (eg LabA1 and LabA2, Figure 2B).10,17 The observation of a 5-fold dehydrated AciA peptide in the in vitro assay indicated an extra dehydration site in addition to the four Dha residues required for labionin formation. β- Mercaptoethanol (βME) was employed to determine the location of the additional Dha residue. After incubating with 5 mM βME at pH 8.5, AciKC-modiﬁed AciA peptide was fully converted to a product containing a single βME adduct, indicating only one dehydroalanine residue was present (Supplementary Figure S6). This result also further conﬁrms the proposed catenulipeptin structure (Figure 2C) because it precludes the possibility of lanthionine formation, which would have resulted in more Dha residues and hence more βME adducts. Subsequent ESI-MS−MS analysis conﬁrmed that the addition of βME occurred at position −1, indicating that Ser−1 was the ﬁfth dehydration site (Supplementary Figure S7). A Dha residue in the leader peptide was also recently reported in another class III lantipeptide erythreapeptin when expressed heterologously.17 The dehydroalanine at the −1 position of modiﬁed AciA oﬀers a convenient strategy to generate mature catenulipeptin in vitro. By reacting with cysteamine under mild alkaline conditions, the Dha−1 was converted into its cysteamine adduct, which is a lysine mimic (Figure 4, Supplementary Figure S8a). Subsequent proteolytic digestion by trypsin resulted in removal of the leader peptide and yielded the mature catenulipeptin in vitro (Figure 4, Supplementary Figure S8b). Articles Figure 4. Generation of catenulipeptin in vitro by AciKC-catalyzed dehydration and cyclization, followed by addition of cysteamine to Dha−1, and treatment with trypsin. With catenulipeptin generated in vitro, we were able to evaluate its bioactivity. The peptide was tested against a number of Gram-positive and Gram-negative bacteria including Escherichia coli DH5α, Bacillus subtilis ATCC6633, Lactococcus lactis HP, and Streptomyces coelicolor; no growth inhibitory activity was observed. Further investigation then focused on possible biological functions of catenulipeptin other than antimicrobial activities. SapB functions as a biological surfactant and facilitates the aerial mycelium formation in its producing strain S. coelicolor.4 The lipopeptide surfactin has been shown to inhibit aerial hyphal formation in S. coelicolor, generating a halo of bald cells that do not have hyphae (Supplementary Figure S9A, right panel).21 SapB can partially restore the aerial growth when applied to surfactin-treated cells. Similarly, when catenulipeptin was applied to wild type S. coelicolor together with surfactin, the diameter of the halo of bald cells was signiﬁcantly reduced (Supplementary Figure S9A, left panel). Although a direct comparison with literature data is diﬃcult, it appears that SapB is more potent than catenulipeptin in this assay (Supplementary Figure S9B). It has also been shown that SapB can trigger early onset of aerial hyphae formation of S. coelicolor.21 When catenulipeptin was applied to plates seeded with a lawn of spores of S. coelicolor A3(2), no acceleration of aerial hyphae formation was observed compared to a control. Furthermore, unlike SapB, which can complement a range of bald mutants (in the bld genes) that cannot generate an aerial mycelium,22 catenulipeptin could not restore aerial hyphae formation in S. coelicolor M600 ΔbldA.23 Thus, whereas catenulipeptin can counteract the balding eﬀect of surfactin, it cannot fully replicate the functionality of SapB on S. coelicolor. Since the molecular mechanism underlying the surfactin- mediated inhibition of aerial hyphae formation is not well 1531 dx.doi.org/10.1021/cb3002446 | ACS Chem. Biol. 2012, 7, 1529−1535