ACS Chemical Biology Articles Figure 1. (A) Structures of labionin and meso-lanthionine and the proposed mechanism of labionin formation by a tandem Michael addition. Shown below the chemical structures are the shorthand notations used throughout this work. (B) Structures of labyrintho- peptin A2 and SapB. function (Figure 2A). Like all other lantipeptide precursor peptides, AciA contains an N-terminal leader peptide that is not modiﬁed and a C-terminal core peptide where the posttransla- tional modiﬁcations take place (vide inf ra).15 A protease for leader peptide removal was not found in the gene cluster nor in neighboring gene clusters. The leader sequence of the peptide substrate AciA contains a Leu-Leu-Asp-Leu-Gln motif (Figure 2B), which is highly conserved in class III lantipeptide precursor peptides and has been shown to be essential for enzymatic processing.16 The core peptide of AciA contains two Ser-Xxx-Xxx-Ser-Xxx-Xxx-(Xxx)-Cys motifs that could be precursors to either lanthionines as in SapB or to labionins as in labyrinthopeptins or both (Figure 2B). Like LabKC and RamC, AciKC consists of an N-terminal lyase domain, a central Ser/Thr kinase domain, and a putative C-terminal cyclase domain based on sequence homology. We ﬁrst examined culture extracts of C. acidiphila DSM 44928 by electrospray ionization (ESI) mass spectrometry (MS) and detected a putative triply charged peptide with a mass of 800.0283 Da (Supplementary Figure S1a). Subsequent tandem MS analysis showed the peptide to be 4-fold dehydrated AciA after removal of a 24 amino acid leader peptide (Supplementary Figure S1b). The fragmentation pattern suggested the formation of two labionins (Figure 2C and Supplementary Figure S1b). Diﬀerent from SapB and labyrinthopeptins, but similar to other recently described class III lantipeptides,17 catenulipeptin possesses a stretch of unmodiﬁed amino acids at its N-terminus. No catenulipeptin Figure 2. (A) Catenulipeptin biosynthetic gene cluster. (B) Sequence alignment of class III lantipeptide precursor peptides. Red lines indicate the proposed lanthionine/labionin rings formed after enzymatic modiﬁcation. RamS is the precursor to SapB. (C) Proposed structure of catenulipeptin. derivatives with overhangs of diﬀerent length were detected by ESI-MS. In Vitro Activity of AciKC. To investigate the biosynthetic pathway, the substrate peptide AciA was expressed in Escherichia coli with an N-terminal hexahistidine tag and three additional lysine residues immediately following the His tag to increase its solubility (His6-K3-AciA). The putative lantipeptide synthetase AciKC was also expressed in E. coli with an N- terminal hexahistidine tag (His6-AciKC). After puriﬁcation (see Supporting Information), His6-K3-AciA was incubated with His6-AciKC in the presence of Mg2+ and ATP. After 4 h, the AciA peptide was dehydrated ﬁve times as determined by matrix-assisted laser desorption ionization (MALDI) MS (Figure 3), which conﬁrmed the dehydratase activity of Figure 3. MALDI-TOF mass spectrum illustrating the in vitro dehydration activity of AciKC. AciA before incubation with AciKC is shown in black, and AciA after incubation with AciKC is shown in red. AciKC. Interestingly, AciKC shows promiscuity with respect to its cosubstrate because use of ATP, GTP, CTP, or TTP all resulted in 5-fold dehydrated AciA peptide (Supplementary Figure S2a−d). However, when NTP was omitted from the assay, no dehydration was observed. The cyclase activity of AciKC was conﬁrmed by incubating the AciKC-modiﬁed AciA peptide with 5 mM iodoacetamide (IAA), a thiol selective reagent, at pH 8.5. This assay did not result in any change in 1530 dx.doi.org/10.1021/cb3002446 | ACS Chem. Biol. 2012, 7, 1529−1535