The secretion of peptides and proteins is essential for survival and ecological adaptation of bacteria. Dual-functional ATP-binding cassette transporters export antimicrobial or quorum signaling peptides in Gram-positive bacteria. Their substrates contain a leader sequence that is excised by an N-terminal peptidase C39 domain at a double Gly motif. We characterized the protease domain (LahT150) of a transporter from a lanthipeptide biosynthetic operon in Lachnospiraceae and demonstrate that this protease can remove the leader peptide from a diverse set of peptides. The 2.0 Å resolution crystal structure of the protease domain in complex with a covalently bound leader peptide demonstrates the basis for substrate recognition across the entire class of such transporters. The structural data also provide a model for understanding the role of leader peptide recognition in the translocation cycle, and the function of degenerate, non-functional C39-like domains (CLD) in substrate recruitment in toxin exporters in Gram-negative bacteria.

The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 Å resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.

Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans. Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii. Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri. Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications. Significance Thioamidation as a posttranslational modification is exceptionally rare, with only one protein example known (methyl-coenzyme M reductase, MCR), as well as a few ribosomal natural products. The genes involved in MCR thioamidation have recently been elucidated, but the enzymes have yet to be characterized. Herein, we report the in vitro reconstitution and substrate preferences of peptidic thioamidation using peptides and enzymes derived from methanogenic archaea. We demonstrate that MCR thioamidation requires an ATP-dependent YcaO enzyme and a sulfide source. Our results shed light on the biosynthesis of other thioamide-containing compounds, which bioinformatics surveys predict to be considerably more numerous than currently appreciated, thus laying a foundation for assigning biological functions for this posttranslational modification.

In several Proteobacteria, LuxI-type enzymes catalyze the biosynthesis of acyl–homoserine lactones (AHL) signals using S-adenosyl–l-methionine and either cellular acyl carrier protein (ACP)-coupled fatty acids or CoA–aryl/acyl moieties as progenitors. Little is known about the molecular mechanism of signal biosynthesis, the basis for substrate specificity, or the rationale for donor specificity for any LuxI member. Here, we present several cocrystal structures of BjaI, a CoA-dependent LuxI homolog that represent views of enzyme complexes that exist along the reaction coordinate of signal synthesis. Complementary biophysical, structure–function, and kinetic analysis define the features that facilitate the unusual acyl conjugation with S-adenosylmethionine (SAM). We also identify the determinant that establishes specificity for the acyl donor and identify residues that are critical for acyl/aryl specificity. These results highlight how a prevalent scaffold has evolved to catalyze quorum signal synthesis and provide a framework for the design of small-molecule antagonists of quorum signaling.

YcaO enzymes are known to catalyze the ATP-dependent formation of azoline heterocycles, thioamides, and (macro)lactamidines on peptide substrates. These enzymes are found in multiple biosynthetic pathways, including those for several different classes of ribosomally synthesized and post-translationally modified peptides (RiPPs). However, there are major knowledge gaps in the mechanistic and structural underpinnings that govern each of the known YcaO-mediated modifications. Here, we present the first structure of any YcaO enzyme bound to its peptide substrate in the active site, specifically that from Methanocaldococcus jannaschii which is involved in the thioamidation of the α-subunit of methyl-coenzyme M reductase (McrA). The structural data are leveraged to identify and test the residues involved in substrate binding and catalysis by site-directed mutagenesis. We also show that thioamide-forming YcaOs can carry out the cyclodehydration of a related peptide substrate, which underscores the mechanistic conservation across the YcaO family and allows for the extrapolation of mechanistic details to azoline-forming YcaOs involved in RiPP biosynthesis. A bioinformatic survey of all YcaOs highlights the diverse sequence space in azoline-forming YcaOs and suggests their early divergence from a common ancestor. The data presented within provide a detailed molecular framework for understanding this family of enzymes, which reconcile several decades of prior data on RiPP cyclodehydratases. These studies also provide the foundational knowledge to impact our mechanistic understanding of additional RiPP biosynthetic classes.