ACS Chemical Biology understood, the role of catenulipeptin in aerial hyphae restoration is not clear and requires further studies. It may potentially be involved in aerial mycelium formation in its producing organism since only one class III lantipeptide gene cluster is present in the genome of C. acidiphila DSM4492813 and this genus has been reported to form aerial hyphae.14 In trans Activity of Core and Leader Peptides. The leader sequences of lantibiotic precursor peptides are important for enzymatic processing but are not strictly required in some examples, such as LctM.24 The possibility that AciKC might also be able to process the AciA core peptide in the absence of the leader sequence was examined by incubation of the core peptide AciA25−51 (1 μM) with AciKC (5 μM). Subsequent MALDI-TOF analysis revealed that no modiﬁcations occurred under such conditions. However, when the leader peptide AciA1−24 was supplied in trans (2 μM), AciKC converted the AciA core peptide into a mixture of phosphorylated and dehydrated products (Supplementary Figure S10). These results clearly show that the leader peptide of AciA, either covalently attached to or supplied in trans with the core peptide, is essential for enzymatic processing of AciKC. Similar to the system of LctA and LctM,25,26 the AciA leader sequence likely activates AciKC by introducing a conformational change. Separation of Dehydration and Cyclization Activity. Intrigued by the mechanism of formation of the unusual labionin structure, we further investigated the cyclization activity of AciKC. A truncated protein, AciKC-ΔC, lacking the C-terminal putative cyclase domain (C-terminal 340 amino acids truncated), retained its dehydration activity (Figure 5A). However, tandem MS−MS analysis revealed that the formation of labionin rings was greatly impaired (Figure 5B, Supple- mentary Figure S11), which conﬁrms the essential role of the C-terminal domain of AciKC for labionin formation. As mentioned previously, the zinc binding amino acid residues are missing in AciKC. To investigate whether perhaps another metal binding site might be present, AciKC was subjected to ICP-MS analysis and PAR assay analysis after removal of loosely bound metal ions by dialysis. Less than 0.1 equiv of zinc was observed by both methods. Furthermore, no signiﬁcant amounts of other metals, such as Co, Fe, and Mg, were detected by ICP-MS. One challenge to investigating multifunctional enzymes is that several reactions take place sequentially in a very short time scale and therefore analysis of one speciﬁc reaction is usually diﬃcult. To further probe the cyclase activity of AciKC, dehydrated peptide substrate was desired. Fortuitously, the dehydratase activity of the AciKC protein tolerates protection of the Cys residues in AciA peptide as an intramolecular disulﬁde. The two cysteine residues in AciA peptide were protected by incubation with oxidized glutathione (Figure 5C). The resulting peptide was then incubated with AciKC, Mg2+, and ATP. Although the disulﬁde bond introduced a cyclic structure into the linear substrate, AciKC dehydrated the peptide with the same eﬃciency as linear AciA. With dehydrated AciA peptide in hand, we were able to investigate whether ATP is required for the cyclase activity of AciKC. Dehydrated AciA peptide (5 μM) was incubated with AciKC (5 μM) in the presence or absence of ATP (5 mM). TCEP (0.1 mM) was added to reduce the disulﬁde bond and release free the Cys thiol groups for cyclization. The samples were incubated for 1 h at room temperature (RT), digested by protease Glu-C, and subsequently analyzed by ESI-MS−MS (Supplementary Figure S12a). The data showed that labionins Articles Figure 5. (A) MALDI-TOF spectrum illustrating the in vitro dehydration activity of AciKC-ΔC. (B) MS−MS fragmentation patterns of unmodiﬁed AciA peptide, AciKC-modiﬁed AciA, and AciKC-ΔC-modiﬁed AciA peptide. (C) Generation of dehydrated AciA peptide with an intramolecular disulﬁde bond and enzymatic cyclization by AciKC in vitro. were formed correctly with or without ATP under such conditions. As a control, AciKC was omitted from the assay and no labionin was formed after incubation of dehydrated AciA and TCEP, as determined by ESI-MS−MS analysis (Supple- mentary Figure S12b). Therefore, ATP is not required for the cyclase activity of AciKC, and nonenzymatic cyclization does not result in labionin formation. As described previously, the leader peptide of AciA is essential for the dehydratase activity of AciKC. Similarly, the leader peptide of LabA2, especially the highly conserved Leu- Leu-Glu-Leu-Gln motif, has been shown to be essential for the dehydratase activity of LabKC.16 However, the role of the leader peptide for the cyclase activity of class III lantipeptide synthetases has not been evaluated yet. Therefore, a truncated 1532 dx.doi.org/10.1021/cb3002446 | ACS Chem. Biol. 2012, 7, 1529−1535