Articles pubs.acs.org/acschemicalbiology Biosynthesis of the Class III Lantipeptide Catenulipeptin Huan Wang and Wilfred A. van der Donk* Howard Hughes Medical Institute and Roger Adams Laboratory, Department of Chemistry, University of Illinois at Urbana−Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, United States *S Supporting Information ABSTRACT: Lantipeptides are ribosomally synthesized and posttranslationally modiﬁed peptides containing lanthionine and/or labionin structures. In this study, a novel class III lantipeptide termed catenulipeptin was discovered from Catenulispora acidiphila DSM 44928, and its biosynthesis was reconstituted in vitro. The multifunctional enzyme AciKC catalyzes both dehydration and cyclization of its peptide substrate AciA and installs two labionin structures in catenulipeptin. AciKC shows promiscuity with respect to cosubstrate and accepts all four NTPs. The C-terminal domain of AciKC is responsible for the labionin formation in catenulipeptin. The cyclase activity of AciKC requires the leader peptide of AciA substrate but does not require ATP or Zn2+. Mutagenesis studies suggest that the labionin cyclization may proceed in a C- to-N-terminal direction. Catenulipeptin partially restores aerial hyphae growth when applied to surfactin-treated Streptomyces coelicolor. L peptides produced by a wide variety of bacteria that are antipeptides are a family of ribosomally synthesized generated by extensive posttranslational modiﬁcations.1,2 Their common structural features are thioether cross-links that are formed between Ser/Thr and Cys residues. The Ser/Thr are ﬁrst dehydrated to dehydroalanine/dehydrobutyrine (Dha/ Dhb), followed by Michael-type addition of Cys residues onto the dehydroamino acids. The resulting thioether structures are called lanthionine (from Ser; Figure 1A) and methyllanthionine (from Thr). Many members of this family of compounds have antimicrobial activities and are therefore named lantibiotics,3 but some lantipeptides exhibit other biological functions. For example, SapB produced by Streptomyces coelicolor (Figure 1B) is a morphogenetic peptide that functions as a biological surfactant and is essential for aerial hyphae formation.4 Lantipeptides have been divided into four classes based on their biosynthetic pathways.5 In compounds from class I, II, and IV, the Michael-type addition is catalyzed by enzymes that contain a catalytic zinc in the active site that is believed to activate the Cys nucleophiles.2,6−8 A domain with sequence homology to these Zn2+-dependent cyclases is also believed to be involved in cyclization of the class III lantipeptides, but this domain does not contain the Zn ligands.4 RamC, the enzyme that generates the lanthionines in SapB by posttranslational modiﬁcation of the RamS peptide, was the ﬁrst class III synthetase identiﬁed.9 Recently, a novel class of lantibiotics was discovered that contain carbocyclic structures called labionins (Figure 1A).10 These structures are formed from two Ser residues and one Cys residue by synthetases that are homologous to RamC. These enzymes contain a central kinase and C-terminal cyclase domain and are therefore called LanKC.10 The kinase domain phosphorylates the Ser and Thr residues to be dehydrated,2,11 and a third N-terminal domain acts as a lyase that eliminates the phosphate from phosphoSer and phosphoThr to generate the Dha and Dhb residues.2,12 The reaction catalyzed by the putative cyclase domain is less well understood. Although RamC and LabKC have 43% sequence identity and their substrate peptides have identical Ser(Xxx)2Ser(Xxx)3Cys motifs, their products, SapB and labyrinthopeptin A2, respectively, are quite diﬀerent (Figure 1B). LabKC installs two labionin motifs in labyrinthopeptin by the addition of a Cys thiol to Dha to generate an enolate intermediate that then adds to a second Dha to form the labionin structure (Figure 1A).11 RamC does not catalyze a tandem addition process, and the initially formed enolates are protonated to generate two typical lanthionine structures (Figure 1B). Intrigued by the novel structures and activities of class III lantipeptides, we decided to investigate one of the other homologues of RamC/LabKC that are encoded in the bacterial genome sequences. In this report, we present a new class III lantipeptide that we termed catenulipeptin and characterize its processing enzyme AciKC. ■ RESULTS AND DISCUSSION Class III Lantipeptide from C. acidiphila DSM 44928. Our study focused on a gene cluster from the actinomycete Catenulispora acidiphila DSM 44928.13,14 Based on sequence homology, this gene cluster encodes a class III lantipeptide synthetase AciKC, a substrate peptide AciA, two ABC transporters, and one hypothetical protein with unknown Received: May 16, 2012 Accepted: June 23, 2012 Published: June 24, 2012 © 2012 American Chemical Society 1529 dx.doi.org/10.1021/cb3002446 | ACS Chem. Biol. 2012, 7, 1529−1535