Unique enzyme chemistry in the biosynthesis of a cyanobacteria neurotoxin April Lukowski 1 , Sanjoy Adak 1 , Rebecca J. B. Schäfer 1 , Bradley S. Moore 1,2 1 Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, USA, 2 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, USA Aetokthonotoxin (AETX) is a potent cyanobacterial neurotoxin responsible for inducing vacuolar myelinopathy in bald eagles and waterfowl in the United States. 1 This small molecule toxin has a remarkable chemical structure with several attractive features, specifically a rare-to-Nature N1-C2' biindole bond, unsymmetrical pentabromination, and a carbonitrile functional group present on only one of the indole monomers. The biosynthetic gene cluster hypothesized to be associated with AETX biosynthesis in the producing organism, Aetokthonoshydrillicola , was identified and consisted of 6 putative enzymes. 1 Upon in vitro characterization of the biosynthetic gene cluster, it was determined that only 5 of the identified genes were required for the complete biosynthesis of AETX. We determined that the biosynthetic pathway follows a convergent route where two functionalized indole monomers are independently generated and reunited by biaryl coupling catalyzed by the cytochrome P450 AetB. Several of the remaining biosynthetic enzymes exhibited impressive catalytic capabilities that would bolster their utility for biocatalytic applications. In particular, we characterized AetF as the first example of a naturally-occurring single-component flavin-dependent tryptophan halogenase and AetD as a novel iron-dependent nitrile synthase that can transform the amino acid portion of tryptophan into a nitrile functional group. This presented work will focus on the characterization of the AETX biosynthetic pathway as well as the ongoing efforts to explore biocatalytic utility and search for homologous gene clusters. References 1. Breinlinger, S.; Phillips, T. J.; Haram, B. N.; Mareš, J.; Martínez, Y. J. A.; Hrouzek, P.; Sobotka, R.; Henderson, W. M.; Schmieder, P.; Williams, S. M.; Lauderdale, J. D.; Wilde, H. D.; Gerrin, W.; Kust, A.; Washington, J. W.; Wagner, C.; Geier, B.; Liebeke, M.; Enke, H.; Niedermeyer, T. H. J.; Wilde, S. B. Hunting the eagle killer: A cyanobacterial neurotoxin causes vacuolar myelinopathy. Science 2021 , 371 , 6536, eaax9050.
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