Exploring the Post-translational Enzymology of PaaA by mRNA Display.

PaaA is a RiPP enzyme that catalyzes the transformation of two glutamic acid residues within a substrate peptide into the bicyclic core of Pantocin A. Here, for the first time, we use mRNA display techniques to understand RiPP enzyme-substrate interactions to illuminate PaaA substrate recognition. Additionally, our data revealed insights into the enzymatic timing of glutamic acid modification. The technique developed is quite sensitive and a significant advancement over current RiPP studies and opens the door to enzyme modified mRNA display libraries for natural product-like inhibitor pans.

Structural Insights into Thioether Bond Formation in the Biosynthesis of Sactipeptides.

Sactipeptides are ribosomally synthesized peptides that contain a characteristic thioether bridge (sactionine bond) that is installed posttranslationally and is absolutely required for their antibiotic activity. Sactipeptide biosynthesis requires a unique family of radical SAM enzymes, which contain multiple [4Fe-4S] clusters, to form the requisite thioether bridge between a cysteine and the α-carbon of an opposing amino acid through radical-based chemistry. Here we present the structure of the sactionine bond-forming enzyme CteB, from Clostridium thermocellum ATCC 27405, with both SAM and an N-terminal fragment of its peptidyl-substrate at 2.04 Å resolution. CteB has the (ß/α)6-TIM barrel fold that is characteristic of radical SAM enzymes, as well as a C-terminal SPASM domain that contains two auxiliary [4Fe-4S] clusters. Importantly, one [4Fe-4S] cluster in the SPASM domain exhibits an open coordination site in absence of peptide substrate, which is coordinated by a peptidyl-cysteine residue in the bound state. The crystal structure of CteB also reveals an accessory N-terminal domain that has high structural similarity to a recently discovered motif present in several enzymes that act on ribosomally synthesized and post-translationally modified peptides (RiPPs), known as a RiPP precursor peptide recognition element (RRE). This crystal structure is the first of a sactionine bond forming enzyme and sheds light on structures and mechanisms of other members of this class such as AlbA or ThnB.

Production of Sactipeptides in Escherichia coli: Probing the Substrate Promiscuity of Subtilosin A Biosynthesis.

Sactipeptides are peptide-derived natural products that are processed by remarkable, radical-mediated cysteine sulfur to α-carbon coupling reactions. The resulting sactionine thioether linkages give rise to the unique defined structures and concomitant biological activities of sactipeptides. An E. coli heterologous expression system, based on the biosynthesis of one such sactipeptide, subtilosin A, is described and this expression system is exploited to probe the promiscuity of the subtilosin A sactionine bond-forming enzyme, AlbA. These efforts allowed the facile expression and isolation of a small library of mutant sactipeptides based on the subtilosin A precursor peptide, demonstrating broad substrate promiscuity where none was previously known. Importantly, we show that the positions of the sactionine linkages can be moved, giving rise to new, unnatural sactipeptide structures. E. coli heterologous expression also allowed incorporation of unnatural amino acids into sactipeptides by means of amber-suppression technology, potentially opening up new chemistry and new applications for unnatural sactipeptides.