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Team:Copenhagen/Project
Overall project
Cytochromes p450:
Cytochromes p450 is one of the largest gene superfamilies coding for enzymes present in the genomes in all biological kingdoms [6]. The enzymatic activities of these proteins are extremely diverse with activity in biotransformation of drugs, bioconversion of xenobiotics, biosynthesis of compounds as steroids, fatty acids, eicosanoids, fat soluble vitamins and bile acids. Furthermore, cytochrome p450’s are involved in the conversion of alkanes, terpenes and aromatic compounds as well as degradation of herbecides and insecticides [6]. We will exploit this activity of cytochromes p450 in the bioconversion of xenobiotics to neutralize damaging medical residues in waste water.
Cytochromes p450 are defined as heme-thiolate proteins featuring a particular spectral signature at 450 nm, thus the name [7]. Despite the label as cytochromes, these proteins are not involved in electron transfers, but act as monooxygenases in a wide range of reactions such as epoxidation, N-dealkylation, O-dealkylation, S-oxidation and hydroxylation [8]. We will focus on the hydroxylating property of the cytochromes, which is also the defining reaction for these enzymes. The reductive activation of molecular oxygen reduces one of the oxygen atoms to a molecule of water, as the other is inserted into the substrate [7]. Fig 1
RH + NAD(P)H + O2 + H+ -> ROH + NAD(P)+ + H2O
A key donor of electrons for the reduction of molecular oxygen is the NADPH dependent cytochrome p450 reductase (CPR). This protein shuttles electrons from NADPH through the FAD and FMN- coenzymes into the iron of the prosthetic heme group of cytochrome p450 [9]. A characterization of the cytochromes p450 is the main objective with this project. We will exploit the wide specificity of these cytochromes to target many different damaging agents continuously by hydroxylation, which can reduce the damaging activity of e.g. estrogen. Fig 1The P450 catalytic cycle in hydroxylation
General experimental design:
We intend to transform E.coli bacteria with different combinations of the several membrane bound human cytochrome p450. Furthermore, our intension is to use different constitutive promoters in order to express the cytochromes at different levels.
The next step will be to examine our different cytochrome p450 transformed E.coli bacteria and their ability to impede estrogenic activity and reduce carbamazepine concentration from water. We hope to find and possibly optimize a biological system which can be introduced in bacteria and lead to the removal of PPCP pollutants in water.
Additional project
Oxime bombs
During our research on Cytochrome p450 another project has begun to form. On the basis of the enclosed Science article “Dynamic Metabolons” by our supervisor, Professor Birger Lindberg Møller, we hope to be able to develop bacteria that can kill fungus. We will introduce a biological system utilizing the plant cytochrome p450 79a1’s ability to produce small molecules called oximes which are toxic to fungus. The idea is quite new and the project is still in progress, but we have very competent supervisors and furthermore they can provide us with most of the parts required. Impact factors:
Open source BioBricks:
Our project has the aim to characterize Cytochrome p450 as a BioBrick and thus add it to the partsregistry of BioBricks, a continuously growing collection of genetic parts that can be mixed and matched to build synthetic biology devices and systems (partsregistry.org). Hence our project contributes to the expansion of the partsregistry, benefitting all researchers with interest in this particular protein, since cytochrome p450 doesn’t exist in the partsregistry. The partsregistry is founded on an open source philosophy, a philosophy we see as a great advantage for development and improvement of the number BioBricks already existing. The addition of cytochrome p450 to this catalogue will perhaps encourage other groups to look at other aspects of the many functions of p450 and thus contribute to the formation of new biological systems – further expanding the field of synthetic biology.
Ecological and economical prospects:
Our future aim with our project is to improve wastewater treatments and thus contribute to a cleaner and uncontaminated drinking water for everyone. With this scientific project, we will move towards this realization, but more research is needed. Our belief is that further research on the use of cytochrome p450 will give a valid and an inexpensive product for use in wastewater treatment plants, thus benefitting the environment and our planet.
