Structural determinants of macrocyclization in substrate-controlled lanthipeptide biosynthetic pathways.

Lanthipeptides are characterized by thioether crosslinks formed by post-translational modifications. The cyclization process that favors a single ring pattern over many other possible ring patterns has been the topic of much speculation. Recent studies suggest that for some systems the cyclization pattern and stereochemistry is determined not by the enzyme, but by the sequence of the precursor peptide. However, the factors that govern the outcome of the cyclization process are not understood. This study presents the three-dimensional structures of seven lanthipeptides determined by nuclear magnetic resonance spectroscopy, including five prochlorosins and the two peptides that make up cytolysin, a virulence factor produced by Enterococcus faecalis that is directly linked to human disease. These peptides were chosen because their substrate sequence determines either the ring pattern (prochlorosins) or the stereochemistry of cyclization (cytolysins). We present the structures of prochlorosins 1.1, 2.1, 2.8, 2.10 and 2.11, the first three-dimensional structures of prochlorosins. Our findings provide insights into the molecular determinants of cyclization as well as why some prochlorosins may be better starting points for library generation than others. The structures of the large and small subunits of the enterococcal cytolysin show that these peptides have long helical stretches, a rare observation for lanthipeptides characterized to date. These helices may explain their pore forming activity and suggest that the small subunit may recognize a molecular target followed by recruitment of the large subunit to span the membrane.

Correction: Structural determinants of macrocyclization in substrate-controlled lanthipeptide biosynthetic pathways.

[This corrects the article DOI: 10.1039/D0SC01651A.].

Assessing the Flexibility of the Prochlorosin 2.8 Scaffold for Bioengineering Applications.

Cyclization is a common strategy to confer proteolytic resistance to peptide scaffolds. Thus, cyclic peptides have been the focus of extensive bioengineering efforts. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a superfamily of peptidic natural products that often contain macrocycles. In the RiPP family of lanthipeptides, macrocyclization is accomplished through formation of thioether cross-links between cysteines and dehydrated serines/threonines. The recent production of lanthipeptide libraries and development of methods to display lanthipeptides on yeast or phage highlights their potential for bioengineering and synthetic biology. In this regard, the prochlorosins are especially promising as the corresponding class II lanthipeptide synthetase ProcM matures numerous precursor peptides with diverse core peptide sequences. To facilitate future bioengineering projects, one of its native substrates, ProcA2.8, was subjected in this study to in-depth mutational analysis to test the limitations of ProcM-mediated cyclization. Alanine scan mutagenesis was performed on all residues within the two rings, and multiple prolines were introduced at various positions. Moreover, mutation, deletion, and insertion of residues in the region linking the two lanthionine rings was tested. Additional residues were also introduced or deleted from either ring, and inversion of ring forming residues was attempted to generate diastereomers. The findings were used for epitope grafting of the RGD integrin binding epitope within prochlorosin 2.8, resulting in a low nanomolar affinity binder of the αvß3 integrin that was more stable toward proteolysis and displayed higher affinity than the linear counterpart.

Insights into AMS/PCAT transporters from biochemical and structural characterization of a double Glycine motif protease.

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.

CylA is a sequence-specific protease involved in toxin biosynthesis.

CylA is a subtilisin-like protein belonging to a recently expanded serine protease family related to class II lanthipeptide biosynthesis. As a leader peptidase, CylA is responsible for maturation of the enterococcal cytolysin, a lantibiotic important for Enterococcus faecalis virulence. In vitro reconstitution of CylA reveals that it accepts both linear and modified cytolysin peptides with a preference for cyclized peptides. Further characterization indicates that CylA activates itself by removing its N-terminal 95 amino acids. CylA achieves sequence-specific traceless cleavage of non-cognate peptides even if they are post-translationally modified, which makes the peptidase a powerful tool for mining novel lanthipeptides by providing a general strategy for leader peptide removal. Knowledge about the substrate specificity of CylA may also facilitate the development of protease inhibitors targeting cytolysin biosynthesis as a potential therapeutic approach for enterococcal infections.

The Enzymology of Prochlorosin Biosynthesis.

Lanthipeptides are ribosomally synthesized and posttranslationally modified peptides containing thioether cross-links formed through addition of a cysteine to a dehydroalanine (to form lanthionine) or to a dehydrobutyrine (to form 3-methyllanthionine). Genome sequencing of marine cyanobacteria lead to the discovery of 1.6 million open reading frames encoding lanthipeptides. In many cases, a genome encodes a single lanthipeptide synthetase, but a large number of substrates. The enzymatic modification process in Prochlorococcus MIT9313 has been reconstituted in vitro, and a variety of experimental approaches have been used to try and understand how one enzyme is capable of modifying 30 different substrates. The methods used to characterize this system will be described along with a brief genomic description of the lanthipeptide landscape found in Prochlorococcus and Synechococcus.

Facile Removal of Leader Peptides from Lanthipeptides by Incorporation of a Hydroxy Acid.

The biosynthesis of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products typically involves a precursor peptide which contains a leader peptide that is important for the modification process, and that is removed in the final step by a protease. Genome mining efforts for new RiPPs are often hampered by the lack of a general method to remove the leader peptides. We describe here the incorporation of hydroxy acids into the precursor peptides in E. coli which results in connection of the leader peptide via an ester linkage that is readily cleaved by simple hydrolysis. We demonstrate the method for two lantibiotics, lacticin 481 and nukacin ISK-1.