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

Authors

Liu, X., Najah, S., Calderari, A., Hong, Z., Halary, S., Lombard, C., Bolard, A., Jeannot, K., Gruez, A., Weissman, K. J., LI, Y.

Abstract

Bacterial azabicyclocarbamates and related pyrrolizidine alkaloids play important roles in microbial interactions, and are scaffolds of therapeutic potential. Their biosynthesis involves a bimodular non-ribosomal 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 core metabolite produced by the major human opportunistic pathogen Pseudomonas aeruginosa, is a rare 4,7-bicyclocarbamate involved in modulating bacterial virulence that belongs to a unique family of natural products targeting ClpP proteases. In this study, we elucidated the full set of reactions leading to the 7-membered cyclocarbamate warhead, and reconstituted in vitro the biosynthesis of azetidomonamide A. Notably, this approach allowed for detailed characterization of a condensation (C) domain-catalyzed online dehydration via chemical capture of NRPS-tethered intermediates. Furthermore, we identified the dehydratase AzeD as the founding member of a distinct group of standalone proteins of the C domain family. Via combined structural, docking and biochemical analyses, we provided evidence that the catalytic mechanism of AzeD is distinct from that of dehydrating C domains, further expanding the known chemistry of these key biosynthetic enzymes.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 19 Jul 2026.

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