Structural investigation of ribosomally synthesized natural products by hypothetical structure enumeration and evaluation using tandem MS

Abstract

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...

Structure and tRNA Specificity of MibB, a Lantibiotic Dehydratase from Actinobacteria Involved in NAI-107 Biosynthesis

“…However in the final natural product Thr12 escapes dehydration (Figure 1B). To eliminate any bias on structural assignments based on the collected fragment ions, tandem MS data were analyzed using an automated hypothetical structure enumeration and evaluation (HSEE) method (Zhang et al, 2014). HSEE serves as an unbiased way for analyzing tandem MS data by matching experimentally observed fragment ions with theoretical fragment ions arising from all possible hypothetical predicted structures (HS).…”

“…We also observed other differences between the two enzymes. NisB dehydrates NisA in a mostly N-to-C terminal fashion (Lubelski et al, 2009, Zhang et al, 2014), whereas MibB processed MibA following a mostly C-to-N terminal directionality after the first dehydration occurred at the N-terminus. Similar order of post-translational modifications that are not strictly directional has been reported for other RiPP biosynthetic enzymes (Jungmann et al, 2014, Melby et al, 2012).…”

New Insights into the Biosynthetic Logic of Ribosomally Synthesized and Post-translationally Modified Peptide Natural Products

“…The fast pace at which bacterial genomes are being sequenced have inspired several research groups to devise automated and semi-automated methods for the structural characterization of novel secondary metabolites in conjunction with the identification of their biosynthetic gene clusters (Doroghazi et al, 2014, Duncan et al, 2015, Kersten et al, 2011, Skinnider et al, 2015, Weber et al, 2015, Zhang et al, 2014). One example of such a pipeline is RiPPquest, which was designed specifically to mine RiPPs (Mohimani et al, 2014).…”

Structure and mechanism of the tRNA-dependent lantibiotic dehydratase NisB

“…The presence of this motif in many class I lantibiotic precursor peptides suggests a similar recognition mechanism employed by their respective lantibiotic dehydratases. In addition, the NisB-catalyzed dehydration of NisA has been shown to proceed with general N -to- C terminal directionality 21,22 . This observation, combined with the demonstration that NisB dehydrates NisA in a distributive manner (Extended Data Fig.…”