Total Biosynthesis of Pseudomonas aeruginosa -Derived Azabicyclocarbamates Identifies Distinct Dehydrating Condensation Family Proteins

Abstract

Bacterial azabicyclocarbamates and related pyrrolizidine alkaloids play important roles in microbial interactions and are scaffolds of therapeutic potential. Their biosynthesis involves a bimodular nonribosomal peptide synthetase (NRPS) as well as a Baeyer–Villiger monooxygenase and tailoring enzymes, the latter contributing to the structural diversification of these compounds. Azetidomonamide A, a rare 4,7-bicyclocarbamate, is a core metabolite produced by the major human opportunistic pathogen Pseudomonas aeruginosa that is involved in virulence modulation. In this study, we elucidate the full set of tailoring reactions leading to azetidomonamide A and reconstitute its biosynthesis in vitro. Notably, this approach allows for detailed characterization of a condensation (C) domain-catalyzed online dehydration via chemical capture of NRPS-tethered intermediates. Furthermore, we identify the dehydratase AzeD as the founding member of a distinct group of standalone proteins of the C domain family. Combined structural, docking and biochemical analyses suggest that the AzeD catalytic mechanism is distinct from that of dehydrating C domains, expanding the known chemistry of these key biosynthetic enzymes. Overall, this work paves the way for accessing these complex molecules and their derivatives by pathway engineering or chemo-enzymatic synthesis.