EAGER: Defining the function and mechanism of YUC1 in tryptophan-dependent indole-3-acetic acid metabolism

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For more than 60 years the question of how plants make the growth hormone auxin has been of major interest to plant biologists. Several issues have created significant confusion including the fact that auxin production at the low levels required for signaling appears to be closely linked to that of several compounds produced in much greater quantities. Auxin is involved in many aspects of plant growth, from early embryo development, to plant stature and form, to root initiation, to fruit development and ripening and plant responses to light, gravity and stress. Improving our understanding of auxin production and the mechanisms for regulation of auxin levels, therefore, has great potential for enhancing both our basic knowledge of these processes and our targeting of genetics for plant improvement. Researchers are now poised to solve the steps for the first pathway for auxin production using a combination of genetic and biochemical approaches. Recent genetic evidence makes it appear that two enzymes, an aminotransferase and a flavin monooxygenase, originally assigned to separate pathways, may together be sufficient for auxin synthesis from tryptophan. To fully define this pathway requires demonstration that the flavin monooxygenase can carry out the novel terminal reaction that has precedent only in enzymes from microbes. This project will test a working hypothesis that the most likely mechanism for the flavin monooxygenase involved in auxin production is a combination of two activities, the oxidative decarboxylation as shown by bacterial lactate monooxygenase and a catalytic cycle using NADPH similar to that of a bacterial phenylacetone monooxygenase. To date, however, no biochemical analysis of the action of the plant flavin monooxygenase protein has been reported on the expected substrate nor is the reaction mechanism known. It is the goal of this EAGER project to elucidate the biochemical reaction carried out by Arabidopsis YUC flavin-monooxygenase, to describe the reaction mechanism, and to determine if the reaction measured in vitro can be shown to occur in vivo.Broader Impacts: This project will support two graduate students and involve the students in both research and scientific writing. These laboratories also serve as a resource in plant metabolomics and to auxin researchers interested in analysis and measurement of hormones at low abundance levels and their metabolism in plants. These laboratories also maintain and encourage ties with several primarily undergraduate institutions, and have been welcome hosts to visitors who require the resources they develop.