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Based on phylogenetic analysis of Arabidopsis thaliana genomic sequences, uncharacterized fatty... mehr
Based on phylogenetic analysis of Arabidopsis thaliana genomic sequences, uncharacterized fatty acid (FA) hydroxylases were identified. Among them were several cytochrome P450s (CYP). Active site model structures of some CYP enzymes revealed that the hydrophobicity in the heme binding site is very different between enzymes active and inactive towards conversion of FAs. Evidence is given that substrate specificity is conserved among phylogenetically related isoforms of CYP enzymes. Particular residues in the active site pocket play key roles in determining substrate specificity. For this thesis metabolite fingerprinting was employed to analyze the corresponding knock-out mutants for the identified genes in order to identify their in vivo substrate specificity. Furthermore heterologous expression of the desired enzymes in yeast or E. coli and affinity-tag dependent purification were the method of choice to investigate the reaction mechanism and substrate specificity of a certain enzyme. To complete these investigations promoter-GUS-constructs and GFP-fusion proteins allowed elucidation of the tissue-specific and sub-cellular localization.