Background: Auxin is essential for plant growth, but its biosynthesis in plants has not been biochemically defined. Results: Key features of the catalytic mechanism for the YUCCA flavoprotein, the rate-limiting enzyme of auxin biosynthesis, are determined. Conclusion: YUCs generate an observable though relatively short lived C4a-(hydro)peroxyflavin intermediate for catalysis in auxin biosynthesis. Significance: This work establishes the previously unknown biochemical mechanism of auxin biosynthesis. Auxin regulates every aspect of plant growth and development. Previous genetic studies demonstrated that YUCCA (YUC) flavin-containing monooxygenases (FMOs) catalyze a rate-limiting step in auxin biosynthesis and that YUCs are essential for many developmental processes. We proposed that YUCs convert indole-3-pyruvate (IPA) to indole-3-acetate (IAA). However, the exact biochemical mechanism of YUCs has remained elusive. Here we present the biochemical characterization of recombinant Arabidopsis YUC6. Expressed in and purified from Escherichia coli, YUC6 contains FAD as a cofactor, which has peaks at 448 nm and 376 nm in the UV-visible spectrum.Weshow that YUC6 usesNADPHand oxygen to convert IPA to IAA. The first step of the YUC6-catalyzed reaction is the reduction of the FAD cofactor to FADH by NADPH. Subsequently, FADH reacts with oxygen to form a flavin-C4a-(hydro) peroxy intermediate, which we show has a maximum absorbance at 381 nm in its UV-visible spectrum. The final chemical step is the reaction of the C4a-intermediate with IPA to produce IAA. Although the sequences of the YUC enzymes are related to those of the mammalian FMOs, which oxygenate nucleophilic substrates, YUC6 oxygenates an electrophilic substrate (IPA). Nevertheless, both classes of enzymes form quasistable C4a-(hydro) peroxylFADintermediates. The YUC6 intermediate has a half-life of μ20 s whereas that of some FMOs is >30 min. This work reveals the catalytic mechanism of the first known plant flavin monooxygenase and provides a foundation for further investigating how YUC activities are regulated in plants.
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