Discovery of a Family of Radical S-Adenosylmethionine Enzymes that Install a Novel Lysine-to-Tryptophan Crosslink

Nature has evolved a wide array of strategies for synthesizing bioactive secondary metabolites. One strategy involves ribosomal biosynthesis of a precursor peptide, which is then modified by one or more tailoring enzymes. The modifications encompass an assortment of cyclization reactions, and the responsible enzymes can often be seen in the biosynthetic gene clusters of the corresponding metabolites. Our lab recently discovered one such cyclic peptide, streptide, which is produced in a quorum sensing-controlled fashion by Streptococcus thermophilus. We showed that streptide contains an unprecedented carbon-carbon crosslink between the β-carbon of a lysine residue and the indole-C7 of a tryptophan side chain. We further demonstrated that the formation of this unusual bond is dependent on a radical S-adenosylmethionine (SAM) metalloenzyme. Herein we show that this enzyme, which we call StrB, contains multiple [4Fe-4S] clusters. One of these clusters binds in the active site and reductively cleaves SAM to generate a 5'-deoxyadenosyl radical, which initiates the radical reaction. Using in vitro kinetic assays coupled with spectroscopic analyses, we establish that StrB installs the novel Lys-to-Trp crosslink in a single step, thus demonstrating a new route for peptide cyclization. On the basis of site-directed mutagenesis of key enzymatic residues and use of substrate analogs containing deuterated amino acids and natural or unnatural amino acid substitutions, we propose a plausible mechanism for this unusual transformation. We further expand the family of Lys-Trp crosslinking enzymes to AgaB and SuiB, StrB homologs encoded in pathogenic Streptococci. Lastly, we report the chemo-enzymatic total synthesis of streptide homologs from these pathogens, which will guide future functional studies of the new family of streptide secondary metabolites

Lysine-Tryptophan-Crosslinked Peptides Produced by Radical SAM Enzymes in Pathogenic Streptococci

Macrocycles represent a common structural framework in many naturally occurring peptides. Several strategies exist for macrocyclization, and the enzymes that incorporate them are of great interest, as they enhance our repertoire for creating complex molecules. We recently discovered a new peptide cyclization reaction involving a crosslink between the side chains of lysine and tryptophan that is installed by a radical SAM enzyme. Herein, we characterize relatives of this metalloenzyme from the pathogens <i>Streptococcus agalactiae</i> and <i>Streptococcus suis</i>. Our results show that the corresponding enzymes, which we call AgaB and SuiB, contain multiple [4Fe-4S] clusters and catalyze Lys-Trp crosslink formation in their respective substrates. Subsequent high-resolution-MS and 2D-NMR analyses located the site of macrocyclization. Moreover, we report that AgaB can accept modified substrates containing natural or unnatural amino acids. Aside from providing insights into the mechanism of this unusual modification, the substrate promiscuity of AgaB may be exploited to create diverse macrocyclic peptides