NRPS amide bond forming domains
Co-complexes between substrate analogues and enzymes can provide excellent insight into substrate specificity and chemical mechanism. Such complexes with the C domain have been elusive because the native substrates rely on transient protein-protein interactions for delivery to the active site and small molecule analogues have low affinities. To overcome this, we developed and used a novel chemical biology approach to capture complexes of substrate analogues bound to the condensation domain. These substrate analogs become covalently tethered near the active site, to mimic covalent substrate delivery by carrier domains. They are competent for reaction and behave similarly to native substrates. This chemical biology approach has enabled determination of co-complexes of the condensation domain for the first time. The complexes strongly suggest that the catalytic histidine’s principal role is to properly position the substrates for catalysis by nucleophilic attack, and allowed insight into the determinants of substrate specificity.
In some NRPS systems, the C domain is replaced by other domains, like an amide-forming transglutaminase domain, or the heterocyclization (Cy) domain. The Cy domain is especially interesting as it not only catalyzes amide bond formation, but also then catalyzes a two-step cyclodehydration between a thiol or hydroxyl side chain and the carbonyl of the newly-formed amide bond, making thiazoline, oxazoline or methyloxazoline rings in the peptide backbone. These heterocyclic rings are important for the bioactivity of peptides such as bacitracin, bleomycin, argyrin, yersiniabactin and colibactin. Strikingly, Cy domains have an extremely conserved DxxxxD motif in the place of the HHxxxDG. Recently, we and Dowling et al. determined the first structures of Cy domains. The structure, biochemistry, bioinformatics and mutational analysis pinpointed new active site residues far in sequences but close in space to the DxxxxD motif that is responsible for the cyclodehydration in Cy domains, allowed proposal of the catalytic mechanisms. Efforts to visualize the Cy domain with substrate and intermediate analogues, which would provide more interesting insight into these multi-functional domains, are underway.
Relevant Schmeing lab papers: Fortinez et al, Nature Comm 2022; Fortinez at al, JACS 2022; Reimer et al, Science 2019; Bloudoff & Schmeing; BBA, 2017; Bloudoff et al, PNAS, 2017; Bloudoff et al, Cell Chem Bio, 2016; Bloudoff et al, JMB, 2013.