Degradation of TCE and EDB

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Wet cleaning” for the aquatic environment

Degradation of TCE and EDB using the CYP2E1 protein in transgenic duckweed

Clean water is essential to the life of all organisms. Because the environment contains toxins and xenobiotics, many organisms have evolved enzymes to degrade such harmful species to less damaging forms. In mammals, cytochrome P450s serve as the primary interface for the metabolism of drugs and other xenobiotics. Human cytochrome P450 2EI (CYP2E1) is one the most conserved xenobiotic metabolizers in mammals1, and it is known for its broad substrate specificity. While it is biologically essential, CYP2E1 is not perfect—due to its mechanism of action; it is hypothesized to uncouple from it accessory proteins, thus allowing reactive intermediates to migrate out of its active site, leading to the damage of tissues2.

Herein we propose a plan to exploit the astounding abilities of CYP2E1. First and foremost, we will remove this protein from its natural environment of the human liver and transform it into common duckweed. Then we will harness it as the central player in a constructed wetland designed to remove xenobiotics directly from contaminated water, rather than the blood stream of a poisoned individual. We will show how human CYP2E1 behaves in the plant system and attempt to correct its known weaknesses.

Specific Aims

Aim 1: Structural characterization and manipulation of human CYP2E1 in complex with plant NADPH-dependent P450 reductase and cytochrome b5

We will use indirect enzyme-linked immunosorbent assays (ELISAs) to determine if human CYP2E1 can bind to plant homologs of NADPH-dependent P450 reductase (NPR) and cytochrome b5 (Cyt b5), then use bimolecular fluorescence complementation (BiFC) to determine the basal interaction of these proteins within Arabidopsis thaliana. Because we plan on enhancing the interaction of human CYP2E1 with the plant accessory proteins, we will first determine if conformational allostery exists within human CYP2E1 and if its association with plant NPR and Cyt b5 affects the substrate-binding pocket. Contact Rearrangement Network (CRN) Analysis will be used to measure changes in the contact between pairs of associating protein crystal structures. Because we hypothesize that we can generate an enzyme that does not uncouple as easily by tightening the interactions between CYP2E1 and its redox partners, we will perform site directed mutagenesis of the amino acids at the interaction interfaces in order to further promote interaction, taking care to avoid the amino acids that are allosterically coupled to the active site. We will perform BiFC assays to determine if the mutants generated interact better. This series of experiments will result in the generation of an enhanced version of CYP2E1, which we will call cytochrome p450 2E1 enhanced or 2E1en.

Aim 2: Transformation of CYP2E1 into duckweed and the characterization of enhanced uptake and metabolism of two common environmental contaminants of the generated system.

We will determine an expression system that best suits 2E1en. Agrobacterium-mediated transformation is commonly used in duckweed3, and it has been used to transform human CYP2E1 into other plants4,

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