RT Journal Article SR Electronic T1 Biological identification and localization of uncyclized xanthommatin, a key intermediate in ommochrome biosynthesis: an in vitro-in vivo study JF bioRxiv FD Cold Spring Harbor Laboratory SP 666529 DO 10.1101/666529 A1 Florent Figon A1 Thibaut Munsch A1 Cécile Croix A1 Marie-Claude Viaud-Massuard A1 Arnaud Lanoue A1 Jérôme Casas YR 2019 UL http://biorxiv.org/content/early/2019/06/11/666529.abstract AB Ommatins are widespread ommochrome pigments mediating important functions in invertebrates. While the early biogenetic steps of ommatins are well known, how their pyrido[3,2-a]phenoxazinone chromophore is formed and modified in vivo remains an unanswered question. In this study, we combined organic synthesis, analytical chemistry and organelle purification to address this issue. We analyzed the metabolites of the tryptophan→ommochrome pathway produced both in vitro and in vivo. We synthesized xanthommatin, the best-known ommatin, by oxidizing 3-hydroxykynurenine in vitro and we analytically characterized the products. We followed the thermal reactivity of synthesized ommatins in acidified methanol to screen for labile and altered compounds. We finally reinvestigated the ommochromes of housefly eyes by purifying and extracting the ommochrome-producing organelles called ommochromasomes. We found that the in vitro oxidative condensation of 3-hydroxykynurenine produces a mixture of ommatins, which readily undergo thermal reactions overtime. Our results suggest that the formation of decarboxylated ommatins might be regulated in vivo, while the methoxylation of ommatins, previously reported in biological extracts, are most likely always artifactitious. For the first time, we identified both in vitro and in vivo the elusive intermediate between 3-hydroxykynurenine and ommatins as being uncyclized xanthommatin. We hence confirm that ommatins are mainly biosynthesized by first the homodimerization of 3-hydroxykynurenine to the aminophenoxazinone uncyclized xanthommatin, and then an intramolecular cyclization to form the pyridine ring. We finally discuss the implication of the newly discovered uncyclized xanthommatin as a branching metabolite in the biosynthesis of ommochromes, particularly of ommins in cephalopods.CEcollision energyCVcone voltageDADdiode-array detectorESI+positive-mode electron spray ionizationLCliquid chromatographyMeOH-HClacidified methanol with 0.5 % hydrochloric acidMRMmultiple reaction monitoringMSmass spectrometryMS/MStandem mass spectrometryMWmolecular weightm/zmass-to-charge ratioNMRnuclear magnetic resonanceRTretention timeSDstandard deviationSEstandard errorSIRsingle ion recordingUVultraviolet