Characterizing the oximidine biosynthetic assembly line for the engineering of novel anticancer agents Eveline Vriens , Dries De Ruysscher, Neeve Marien, Evi Gorrens, Joleen Masschelein KU Leuven - VIB Center for Microbiology, Belgium Vacuolar H + -ATPases (V-ATPases) are ATP-driven proton pumps that carefully control the pH homeostasis in eukaryotic intracellular compartments to enable essential cellular processes, such as protein degradation and membrane trafficking. Their malfunctioning is associated with a range of human diseases, including cancer, osteoporosis and neurodegeneration. This, together with the fact that they occur in many different isoforms, makes V-ATPases attractive and potentially specific drug targets 1 .One class of specific V-ATPases inhibitors are the oximidines 2 . These benzolactone enamides are produced by Pseudomonas baetica and show potent and selective cytotoxicity towards cancer cells.Interestingly, the hybrid polyketide synthase non-ribosomal peptide synthetase assembly line responsible for the biosynthesis of the oximidines contains several non-canonical features. Firstly, the oximidines contain a rare O-methyloxime moiety which is believed to be installed on the assembly line by the concerted action of a monooxygenase, tautomerase and methyltransferase domain. Secondly, after chain release, a putative cytochrome P450 enzyme introduces a varying range of oxidative modifications, such as epoxidation and hydroxylation, resulting in diverse oximidine variants. Here, we used a combination of in vitro biochemicals assays with purified recombinant enzymes and chemically-synthesized substrate mimics, along with in vivo mutagenesis approaches to investigate these unusual enzymes and elucidate their mechanism of action and substrate specificity. Together, our results may pave the way for rational pathway engineering strategies to enhance the biological activity of the oximidines. References 1. Collins, M. P. and Forgac, M. Biochim. Biophys. Acta Biomembr. 2020, 1862 , 183341 2. Pérez-Sayáns, M., Somoza-Martín, J. M., Barros-Angueira F., Gándara Rey, J.M. and García-García, A. Cancer Treatment Reviews .2009, 35 , 707–713
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