Project (Ivo Feussner)
Non-targeted ex vivo metabolomics for functional gene annotation
Powerful improvements in sequencing technologies rapidly increase the number of sequenced genomes, but still unequivocally functional annotation of genes is the bottleneck to make genome data suitable for biological interpretation, e.g. understanding of metabolism and signalling cascades. Although comparative sequence analysis can provide first hints in respect to the expected reaction catalyzed by a gene product of interest, in vitro studies with purified enzymes are necessary to confirm the activity and the respective substrate(s). However, the complete plethora of endogenous substrates, which are physiologically available in a cell cannot be covered by this targeted approach. To close this gap, we developed a method to bring the heterologous expressed and purified enzyme back to its complex native metabolite environment (total metabolome extracts of the respective tissue and the indicated developmental stage etc.). Further, we developed a comprehensive non-targeted metabolomics approach by UPLC TOF-MS, which allows us to identify biological relevant substrates and the corresponding products within a complex biological matrix (Kaever, et al. 2015). The large data sets are processed (ranking, filtering, adduct correction, data merging, database search) and visualized with help of our software suite MarVis (http://marvis.gobics.de/). The project will deal with the enzyme class of GH3 amino acid conjugases in plants. It is known, that GH3 enzymes are necessary for the regulation of stress-induced hormone metabolism and plant defence (Okrent et al. 2011). The aim of the work is to unravel the complete substrate specificity of all 19 GH-enzymes of this class for functional analysis and to describe their interplay in the hormone homeostasis. Work on this project requires talented experimentalists with strong interest and experience in heterologous expression and purification of proteins as well as in analytical methods and computational science.
Kaever A, Landesfeind M, Feussner K, Mosblech A, Heilmann I, et al. 2015. MarVis-Pathway: integrative and exploratory pathway analysis of non-targeted metabolomics data. Metabolomics 11:764-77
Okrent RA, Wildermuth MC. 2011. Evolutionary history of the GH3 family of acyl adenylases in rosids. Plant Mol Biol 76:489-505
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