The lantibiotic nisin is an antimicrobial peptide that is widely used as a food preservative to combat food-borne pathogens1. Nisin contains dehydroalanine and dehydrobutyrine residues that are formed via dehydration of Ser/Thr by the lantibiotic dehydratase NisB2. Recent biochemical studies revealed that NisB glutamylates Ser/Thr side chains as part of the dehydration process3. However, the molecular mechanism by which NisB utilizes glutamate to catalyze dehydration remains unresolved. Here we show that this process involves glutamyl-tRNAGlu to activate Ser/Thr residues. In addition, the 2.9 Å crystal structure of NisB in complex with its substrate peptide NisA reveals the presence of two separate domains that catalyze the Ser/Thr glutamylation and glutamate elimination steps. The co-crystal structure also provides the first insights into substrate recognition by lantibiotic dehydratases. Our findings demonstrate a non-anticipated role for aminoacyl-tRNA in the formation of dehydroamino acids in lantibiotics, and serve as a basis for the functional characterization of the many lantibiotic-like dehydratases involved in the biosynthesis of other classes of natural products.

Summary Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large group of structurally diverse natural products. Their biological activities and unique biosynthetic pathways have sparked a growing interest in RiPPs. Furthermore, the relatively low genetic complexity associated with RiPP biosynthesis makes them excellent candidates for synthetic biology applications. This review highlights recent developments in the understanding of the biosynthesis of several bacterial RiPP family members, the use of the RiPP biosynthetic machinery for generating novel macrocyclic peptides, and the implementation of tools designed to guide the discovery and characterization of novel RiPPs.

Summary Class I lantibiotic dehydratases dehydrate selected Ser/Thr residues of a precursor peptide. Recent studies demonstrated the requirement of glutamyl-tRNAGlu for Ser/Thr activation by one of these enzymes (NisB) from the Firmicute Lactococcus lactis. However, the generality of glutamyl-tRNAGlu usage and the tRNA specificity of lantibiotic dehydratases have not been established. Here we report the 2.7-Å resolution crystal structure, along with the glutamyl-tRNAGlu utilization of MibB, a lantibiotic dehydratase from the Actinobacterium Microbispora sp. 107891 involved in the biosynthesis of the clinical candidate NAI-107. Biochemical assays revealed nucleotides A73 and U72 within the tRNAGlu acceptor stem to be important for MibB glutamyl-tRNAGlu usage. Using this knowledge, an expression system for the production of NAI-107 analogs in Escherichia coli was developed overcoming the inability of MibB to utilize E. coli tRNAGlu. Our work provides evidence for a common tRNAGlu-dependent dehydration mechanism, paving the way for the characterization of lantibiotics from various phyla.

Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides containing thioether rings. In addition to these crosslinks, the clinical candidate lantibiotic NAI-107 also possesses a C-terminal S-[(Z)-2-aminovinyl]-D-cysteine (AviCys) and a unique 5-chloro-L-tryptophan (ClTrp) moiety linked to its potent bioactivity. Bioinformatic and genetic analyses on the NAI-107 biosynthetic gene cluster identified mibH and mibD encoding flavoenzymes responsible for the formation of ClTrp and AviCys, respectively. The biochemical basis for the installation of these modifications on NAI-107 and the substrate specificity of either enzyme is currently unknown. Using a combination of mass spectrometry, liquid chromatography, and bioinformatic analyses we demonstrate that MibD is an FAD-dependent Cys decarboxylase and that MibH is an FADH2-dependent Trp halogenase. Most FADH2-dependent Trp halogenases halogenate free Trp, but MibH was only active when Trp was embedded within its cognate peptide substrate deschloro NAI-107. Structural comparison of the 1.85-Å resolution crystal structure of MibH with other flavin-dependent Trp halogenases revealed that subtle amino acid differences within the MibH substrate binding site generates a solvent exposed crevice presumably involved in determining its unusual substrate specificity.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a growing class of natural products that are found in all domains of life. These compounds possess vast structural diversity and have a wide range of biological activities, promising a fertile ground for exploring novel natural products. One challenging aspect of RiPP research is the difficulty of structure determination due to their architectural complexity. We here describe a method for automated structural characterization of RiPPs by tandem mass spectrometry. This method is based on the combined analysis of multiple mass spectra and evaluation of a collection of hypothetical structures predicted based on the biosynthetic gene cluster and molecular weight. We show that this method is effective in structural characterization of complex RiPPs, including lanthipeptides, glycopeptides, and azole-containing peptides. Using this method, we have determined the structure of a previously structurally uncharacterized lanthipeptide, prochlorosin 1.2, and investigated the order of the posttranslational modifications in three biosynthetic systems.dehydration | genome mining | lantibiotics | directionality R ibosomally synthesized and posttranslationally modified peptides (RiPPs) are a major class of natural products as revealed by the genome-sequencing efforts of the past decade (1). RiPPs are biosynthesized from genetically encoded and ribosomally produced precursor peptides, which typically consist of a core peptide that is transformed to the final product and an N-terminal extension called the leader peptide that is usually important for recognition by the posttranslational modification (PTM) enzymes (1). Because of the highly diverse PTMs, these compounds possess vast structural diversity and have a wide range of biological activities, thus representing a fertile ground for exploration. Furthermore, the ribosomal origin of RiPPs makes them particularly well suited for genome mining efforts. By using genome mining to explicitly avoid species harboring biosynthetic gene clusters identical to those that produce known compounds, a combination of strain prioritization and mass spectrometry (MS)-based analysis offers a new route to discovering natural products that can overcome the burden of rediscovery that has increasingly hampered discovery efforts (2, 3). One challenging aspect of high-throughput genome mining for new natural products is the difficulty to determine their molecular structures in high throughput. We present here a method that allows automated RiPP structure elucidation.In contrast to nonribosomal peptides that have an average molecular weight of less than 1,000 Da, as documented in the NORINE database (4), RiPPs in many cases have molecular weights larger than 2,500 Da. Molecules of this size are difficult to rapidly analyze by NMR spectroscopy, rendering MS the most convenient tool for RiPP structural characterization. Even when the precursor peptide sequences are known and the types of PTMs can be predicted based on the sequen...