Biosynthesis of the unusual carbon skeleton of nocuolin A Teresa Patricia Martins 2 and Teresa Patricia Pedrosa Martins 1, Nathaniel R. Glasser 3 , Duncan J. Kountz 3 , Paulo Oliveira 4,5,6 , Emily P. Balskus 3* , Pedro N. Leão 1* 1 CIIMAR, Portugal, 2 ICBAS, Portugal, 3 Harvard University, USA, 4 i3S, University of Porto, Portugal, 5 IBMC,University of Porto, Portugal, 6 Biology Department, University of Porto, Portugal Natural products are useful for their biological activities, but their intricate structures also intrigue and inspire biochemists to discover new biosynthetic reactions. Nocuolin A is a cytotoxic compound produced by cyanobacteria and predicted to be the product of the noc pathway 1 . Notably, nocuolin A features a 1,2,3-oxadiazine moiety, unique among natural products (and so far inaccessible through organic synthesis), suggesting that novel enzymatic chemistry might be involved in its biosynthesis. We report here our findings on the origin of the carbon skeleton of the nocuolin A. Supplementation experiments with stable isotope-labeled fatty-acids of varying chain lengths revealed that the C 13 chain is assembled from two short-chain fatty acids, hexanoic and octanoic acids. We confirmed these observations through enzymatic assays carried out with purified noc biosynthetic pathway enzymes. Specifically, we show that a fatty-acyl AMP ligase activates both hexanoic and octanoic acids which undergo a posterior non-decarboxylative Claisen condensation catalyzed by a new ketosynthase. This new class of enzymes is mainly found in Cyanobacteria and Actinobacteria and extends the diversity of C-C bond forming enzymes known to date. Further supplementation experiments with stable isotope-labeled methionine indicated that the 3-hydroxypropanoyl moiety originates from this amino acid, through a yet-unrecognized mechanism. With this work we obtain the first biochemical evidence connecting the putative noc pathway to nocuolin A, dissect the biosynthesis of the C 13 carbon skeleton and characterize a new class of ketosynthases. We also obtain evidence for unusual biochemistry leading to the 3-hydroxypropanoyl moiety, which will be the subject of future studies. References 1. Voracova, K.; Hajek, J.; Mares, J.; Urajova, P.; Kuzma, M.; Cheel, J.; Villunger, A.; Kapuscik, A.; Bally, M.; Novak, P.; Kabelac, M.; Krumschnabel, G.; Lukes, M.; Voloshko, L.; Kopecky, J.; Hrouzek, P., The cyanobacterial metabolite nocuolin a is a natural oxadiazine that triggers apoptosis in human cancer cells. PLoS One 2017, 12 (3), e0172850.
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