Team:Copenhagen/Project

CyperMan

The world of today faces numerous environmental problems - man-made and natural occuring alike and ranging from water to air. We will introduce CyperMan to this suffering world. The CyperMan is a bacteria that utillerizes the family of cytochrome p450s (CYPs) for bioremediation. CYPs are found in countless species and come in many forms - each with unique attributes but sharing a commen mechanism. We see great potential for CyperMans and we believe that they can be used for several causes. Our project is designed to demonstrate this diversity as our two CYPs combats environmental problems in water as well as on land - with the same enzyme.
CyperMan - Saves the Fish

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 water. We hope to find and possibly optimize a biological system which can be introduced in bacteria and lead to the removal of PPCP (pharmaceutical and personal care products) pollutants in water.

CyperMan - Kills fungus

We hope to be able to develop bacteria that can kill fungus. We will introduce a biological system to ''E.coli'' utilizing the plant cytochrome p450 79’s ability to produce small molecules called oximes which are toxic to fungus.

Cytochrome 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.

Figure 1The P450 catalytic cycle in hydroxylation