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.DOI: http://dx.doi.org/10.7554/eLife.07607.001

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.

Beta-hairpins constitute the smallest beta-type structures in peptides and proteins. The development of highly stable, yet monomeric beta-hairpins based on the tryptophan zipper motif was therefore a remarkable success [A. G. Cochran, N. J. Skelton, M. A. Starovasnik, Proc. Natl. Acad. Sci USA 2001, 98, 5578-5583]. We have been able to design, synthesize and characterize a hairpin based on this motif which incorporates an azobenzene-based photoswitch, that allows for time-resolved folding studies of beta-structures with unprecedented time resolution. At room temperature the trans-azo isomer exhibits a mostly disordered structure; however, light-induced isomerization to the cis-azo form leads to a predominantly extended and parallel conformation of the two peptide parts, which are linked by the novel photoswitch, [3-(3-aminomethyl)phenylazo]phenylacetic acid (AMPP). While in the original sequence the dipeptide Asn-Gly forms a type I' beta-turn which connects the two strands of the hairpin, this role is adopted by the AMPP chromophore in our photoresponsive beta-hairpin that can apparently act as a beta I'-turn mimetic. The beta-hairpin structure was determined and confirmed by NMR spectroscopy, but the folding process can be monitored by pronounced changes in the CD, IR and fluorescence spectra. Finally, incorporation of the structurally and functionally important beta-hairpin motif into proteins by chemical ligation might allow for the photocontrol of protein structures and/or functions.

A light-switchable peptide is transformed with ultrashort pulses from a ␤-hairpin to an unfolded hydrophobic cluster and vice versa. The structural changes are monitored by mid-IR probing. Instantaneous normal mode analysis with a Hamiltonian combining density functional theory with molecular mechanics is used to interpret the absorption transients. Illumination of the ␤-hairpin state triggers an unfolding reaction that visits several intermediates and reaches the unfolded state within a few nanoseconds. In this unfolding reaction to the equilibrium hydrophobic cluster conformation, the system does not meet significant barriers on the free-energy surface. The reverse folding process takes much longer because it occurs on the time scale of 30 s. The folded state has a defined structure, and its formation requires an extended search for the correct hydrogen-bond pattern of the ␤-strand.density functional theory calculation ͉ peptide folding ͉ TrpZip2 ͉ ultrafast infrared spectroscopy F olding is a key process during the formation of a functional protein after the synthesis of its amino acid chain (1). During folding, the amino acid chains are rearranged in highly complex processes, for which a detailed understanding is still missing. Straightforward solutions of the folding problem are prevented by the high dimensionality of the protein conformational spaces and by the wide range of relevant time scales. Thus, for complexity reduction, one has to focus on typical protein substructures, which are small enough to be analyzed by present-day technology. With corresponding peptide model systems there is a chance to monitor the formation of secondary structures, to identify characteristic intermediate states of these folding dynamics, and to carry out realistic simulations on a molecular level. An interesting class of model systems are light-triggered peptides, within which the incorporation of a photoresponsive element can initiate structural changes.Among corresponding photoresponsive chromophores, azobenzene derivatives have become a popular choice when it comes to selecting a fast conformational trigger for peptide refolding, because their structure significantly changes on time scales of a few hundred femtoseconds after photoexcitation. Indeed, an azobenzene chromophore, used as a backbone element within cyclic peptides, was shown to induce large-scale conformational changes, whose dynamics could be monitored by visible (2) and IR (3) spectroscopy. Moreover, the experimental results clearly showed that the initial, strongly driven conformational changes of the peptide proceeded within a few picoseconds after the trigger event. Externally linked to an ␣-helix, azobenzene also was used for unfolding (4) and refolding (5) of an ␣-helical model peptide in the nanosecond to microsecond time range.A prominent secondary structure motif is the ␤-sheet, for which a ␤-hairpin represents a minimal model (6, 7). We and others (8-10) have recently focused on the design of photocontrolled ␤-hairpin peptides. To enable an ultrafa...

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 and genes of 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 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 and 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 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-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.

Here we report a transcription factor decoy strategy for targeted activation of eight large silent polyketide synthase and non-ribosomal peptide synthetase gene clusters, ranging from 50 to 134 kilobases in multiple streptomycetes, and characterization of a novel oxazole family compound produced by a 98-kb biosynthetic gene cluster. Due to its simplicity and ease, this strategy can be readily scaled up for discovery of natural products in streptomycetes.

In several , LuxI-type enzymes catalyze the biosynthesis of acyl-homoserine lactones (AHL) signals using-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 -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.