Sassi, G.; Mori, G.; Facchetti, S.; Alvarenga, J. F. R. d.; Robescu, M. S.; Dembech, E.; Vezzoli, M.; Merici, G.; Malatesta, M.; Dobani, S.; Rio, D. D.; Bresciani, L.; Quilici, G.; Cavazzini, D.; Battistutta, R.; Sansone, F.; Armirotti, A.; Ferrari, R.; Mena, P.; Ubiali, D.; Peracchi, A.; Rivetti, C.; Percudani, R.

Most tryptophan catabolism in animals occurs through the kynurenine pathway, which generates the essential NAD cofactor and multiple bioactive metabolites. Knowledge of this pathway in eukaryotes ends at the unstable intermediate 2-aminomuconate (2-AM). Here, by leveraging evolutionary information from more than 5,000 eukaryotes, we identify two distinct genes acting downstream of 2-AM in fungi and metazoa. The fungal gene is homologous to bacterial 2-AM deaminase, whereas the metazoan gene is homologous to aspartate dehydrogenase (ASPDH), which in prokaryotes catalyses the first reaction of NAD biosynthesis. Biochemical and structural analyses show that human ASPDH has evolved an unprecedented function as an NAD(P)H-dependent 2-AM reductase (AMR) in tryptophan catabolism. The reaction forms L-2-aminohex-3-enedioic acid, an unsaturated -amino acid absent from current biological databases. Isotope-labeling NMR experiments and structural modelling support a mechanism in which hydride transfer is coupled to double-bond rearrangement of the conjugated system. These findings reveal a previously unknown metazoan branch of the kynurenine pathway, expand the repertoire of endogenous amino acids, and illustrate how comparative genomics can uncover hidden reactions in human metabolism.