Regulation and functionality of stress-induced cytochrome P450 monooxygenases: A functional genomics approach

The current progress of genomic research has led to the identification of appreciable numbers of plant cytochrome P450 monooxygenase enzymes, which have been found to play intimate roles in important processes such as plant defense, viability and development. A detailed understanding of the physiological roles and impacts on plant development or regulatory processes is essential before utilizing these enzymes for agricultural purposes, as in engineering more suitable and sustainable crops. Investigations on the regulation and functionalities of proteins were performed towards functional definition of stress-induced cytochrome P450 monooxygenases (P450s) from Arabidopsis thaliana, a model plant system.; Two P450 enzymes, CYP94B1 and CYP94C1, were selected for functional analyses based on microarray data that demonstrates their transcriptional inducibility in plants under conditions representative of wounding, insect attack, drought, or osmotic stresses. Conventional methods for evaluating protein sequence, structure and function were used to select and study the Arabidopsis P450 enzymes. Analyses of primary sequences led to the prediction of cellular locations and identification of amino acid conservations. Sequence comparisons to P450s in other plant species and spectral properties intrinsic to this enzyme superfamily were exploited to identify a range of potential substrates. Using these approaches, members of the CYP94B and CYP94C family were found to have fatty acid hydroxylase activity.; Whereas Arabidopsis P450s are typically membrane-associated, the hydrophobic nature of membrane-binding regions oftentimes creates difficulties in the solubilization, isolation and study of these proteins using traditional biochemical techniques. Paralleling the study of individual P450s was the development of a biotechnique to transfer membrane proteins directly from partially fractionated cellular membranes into soluble nanostructures that contain a native-like membrane environment. The resulting system is conducible to many of the biochemical methodologies previously restricted to soluble proteins and adds another tool to aid in the characterizations of this important class of enzymes.