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

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

doi:10.64898/2026.07.17.738954

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bioRxiv · 10.64898/2026.07.17.738954

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

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

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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 AzeDs catalytic mechanism is distinct from that of dehydrating C domains, further expanding the known chemistry of these key biosynthetic enzymes.

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Liu, X., Najah, S., Calderari, A., Hong, Z., Halary, S., Lombard, C., Bolard, A., Jeannot, K., Gruez, A., Weissman, K. J., LI, Y.. 2026-07-17. Total Biosynthesis of Pseudomonas aeruginosa-Derived Azabicyclocarbamates Identifies Distinct Dehydrating Condensation Family Proteins. https://doi.org/10.64898/2026.07.17.738954

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