Characterization of a Dehydratase and Methyltransferase in the Biosynthesis of Ribosomally Synthesized and Post‐translationally Modified Peptides in Lachnospiraceae

Abstract

As a result of the exponential increase in genomic data, discovery of novel ribosomally synthesized and post‐translationally modified peptide natural products (RiPPs) has progressed rapidly in the past decade. The lanthipeptides are a major subset of RiPPs. Through genome mining we identified a novel lanthipeptide biosynthetic gene cluster (lah) from Lachnospiraceae bacterium C6A11, an anaerobic bacterium that is a member of the human microbiota and which is implicated in the development of host disease states such as type 2 diabetes and resistance to Clostridium difficile colonization. The lah cluster encodes at least seven putative precursor peptides and multiple post‐translational modification (PTM) enzymes. Two unusual class II lanthipeptide synthetases LahM1/M2 and a substrate‐tolerant S‐adenosyl‐l‐methionine (SAM)‐dependent methyltransferase LahSB are biochemically characterized in this study. We also present the crystal structure of LahSB in complex with product S‐adenosylhomocysteine. This study sets the stage for further exploration of the final products of the lah pathway as well as their potential physiological functions in human/animal gut microbiota.

Biosynthesis and Heterologous Expression of Cacaoidin, the First Member of the Lanthidin Family of RiPPs

“…No other CypM or LxmM homologues have been found in the genome of strain CA-170360. Cao4 shows very low homology with two types of LanS O-methyltransferases described in class I lanthipeptide clusters from actinobacteria: LanS A [ 26 ] and LanS B [ 27 ]. Thus, its role in the N , N -methylation remains unclear and is currently under study.…”

“…Thus, its role in the N,N-methylation remains unclear and is currently under study. D-Amino acids provide a wide variety of properties to lanthipeptides [28] and are introduced by modifying the genetically encoded L-Ser and L-Thr into Dha and Dhb, which are subjected to a diastereoselective hydrogenation to finally incorporate D-Ala and D-Abu, respectively [15,27]. This reaction is carried out by dehydrogenases generically called LanJ [15], which are divided into zinc-dependent dehydrogenases (LanJ A ), which can only hydrogenate Dha, and flavin-dependent dehydrogenases (LanJ B ) able to reduce both Dha and Dhb [29,30].…”

New developments in RiPP discovery, enzymology and engineering

“…Nucleotide binding results in a rotation of the NBDs, which closes the transmembrane tunnel and displaces the peptidase domains. 305 Investigation of the substrate specicity and recognition mechanism of the N-terminal peptidase domain of LahT (LahT150), the PCAT associated with a currently uncharacterized RiPP (Section 5.3.7.1), 307 demonstrated tolerance to many substrates. 308 The key residues that are important for LP binding are Val(À4), Leu(À7), and Leu(À12), which are Fig.…”

“…Its activity was LP independent and a crystal structure was reported for the enzyme. 307 The nal structures of the RiPPs produced by this BGC are currently not known. In contrast to C-terminal carboxylate methylation, the carboxylate side chain of D-Asp in bottromycin is methylated by BmbA (Fig.…”

Transporter Protein-Guided Genome Mining for Head-to-Tail Cyclized Bacteriocins

“…With the rapid advances in DNA sequencing technologies and the development of bioinformatic tools, a plethora of genomic data can now be mined for biosynthetic gene clusters that potentially encode novel natural products, both ribosomal and non-ribosomal in origin [ 22 , 23 , 24 , 25 , 26 , 27 ]. In 2017, the head-to-tail cyclized bacteriocin pumilarin from Bacillus pumilus B4107 was discovered through genome mining with the use of the bioinformatics tool BAGEL3 wherein the authors filtered their candidate biosynthetic gene clusters based on the presence of genes encoding a precursor peptide and a SpoIIM protein [ 28 , 29 ].…”