Team:Copenhagen/Project/Water

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<strong>Cytochrome p450 CYP 1 & 2 - the source of CyperMans water cleansing power</strong>
 
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Cytochromes p450 is one of the largest gene superfamilies coding for enzymes present in the genomes in all biological kingdoms [1]. 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 [1]. </p>
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<p>We will exploit this activity of cytochromes p450 in the bioconversion of xenobiotics to neutralize damaging medical residues in waste water.</p>
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<b><center>The source of CyperMans <br><br>water cleansing power</center></b></font>
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<p>Cytochromes p450 are defined as heme-thiolate proteins featuring a particular spectral signature at 450 nm, thus the name [2]. 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 [3]. </p>
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<p>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 [2].</p>
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Fig 1
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<b><center>Cytochrome p450 CYP 1 and 2</center></b></font>
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<strong>RH + NAD(P)H + O2 + H+ -> ROH + NAD(P)+ + H2O</strong>
 
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<b>Background information:</b>
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<p>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 that it excerts when it binds to the estrogen receptors to often.  
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<p>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 [4]. </p>
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Our solution to that problem is to change the estrogen into a compound that has a low affinity for the estrogen receptors. 2-hydroxyestradiol with an affinity for the estrogen receptor at about 25% of estrogen is such a compound(1). The CYPs we'll explore are CYP1A1, CYP1A2, CYP2C9 and CYP3A4.
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<p>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. </p>
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The human isoforms CYP1A2 has been proven to have a very high catalytic activity for hydroxylation of the number 2 carbon of estradiol in addition to a lower catalytic activity for the number 4 carbon(2). In addition it has been shown that CYP2C9 has a very specific catalytic activity this carbon as well(2). The mechanism that the CYP's use is shown below
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<table class="https://static.igem.org/mediawiki/2011/c/cc/CYP_catalystisk_circle.PNG" align="center">
<table class="https://static.igem.org/mediawiki/2011/c/cc/CYP_catalystisk_circle.PNG" align="center">
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  <caption align="bottom"><p align="justify"><b>Figure 1</b>The P450 catalytic cycle in hydroxylation</p></caption>
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  <caption align="bottom"><p align="center"><b> The P450 catalytic cycle in hydroxylation</b></p></caption>
<tr><td><img src="https://static.igem.org/mediawiki/2011/c/cc/CYP_catalystisk_circle.PNG" width="500px"></td></tr>
<tr><td><img src="https://static.igem.org/mediawiki/2011/c/cc/CYP_catalystisk_circle.PNG" width="500px"></td></tr>
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<b>The project:</b>
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<u>Ecological and economical prospects:</u>
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Another 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.
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It is our intention to insert a plasmid into <i>E. Coli</i> containing a promoter that we control as well as the sequence for one of the CYP's. After that we will turn our bacteria into cytochrome p450 factories in order to create enough so that they can convert a lot of 17beta-estradiol into 2hydroxyestradiol.
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As a courtesy of Quo Data we have been granted a A-Yes-Assay kit, that allows us to test how many compounds in water that reacts with the estrogen receptors. Hopefully if our CYP's are effective, we will observe that the ammount of compounds that react with the receptors will decrease.
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<strong>References</strong>
 
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[1] F. Hannemann, A. Bichet, K. M. Ewen, R. Bernhardt: Cytochrome p450 systems – biological variations of electron transport chains. Biochemica et Biophysica Acta 1770 (2007) 330 - 344
 
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[2] Paul R. Ortiz de Montellano. Hydrocarbon Hydroxylation by Cytochrome P450 Enzymes. Chem Rev. 2010 February 10; 110(2): 932
 
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[3] http://www.anaesthetist.com/physiol/basics/metabol/cyp/Findex.htm
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<u>Ecological and economical prospects:</u>
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[4] T. Laursen, K. Jensen,B. L. Møller: Conformational changes of the NADPH dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450. Biochemica et Biophysica Acta 1814 (2011) 132-138
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Another 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.
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<u>References:</u><br>
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1. Schutze, N., Vollmer, G., Wunsche, W., Grote, A., Feit, B., and Knuppen, R. (1994) Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to the estrogen receptor of MCF-7 cells in cytosolic extracts and in nuclei of intact cells, Exp Clin Endocrinol 102, 399-408.<br>
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2. Lee, A. J., Cai, M. X., Thomas, P. E., Conney, A. H., and Zhu, B. T. (2003) Characterization of the oxidative metabolites of 17beta-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms, Endocrinology 144, 3382-3398.
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Latest revision as of 21:14, 21 September 2011



The source of CyperMans

water cleansing power



Cytochrome p450 CYP 1 and 2
Background information:

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 that it excerts when it binds to the estrogen receptors to often. Our solution to that problem is to change the estrogen into a compound that has a low affinity for the estrogen receptors. 2-hydroxyestradiol with an affinity for the estrogen receptor at about 25% of estrogen is such a compound(1). The CYPs we'll explore are CYP1A1, CYP1A2, CYP2C9 and CYP3A4.
The human isoforms CYP1A2 has been proven to have a very high catalytic activity for hydroxylation of the number 2 carbon of estradiol in addition to a lower catalytic activity for the number 4 carbon(2). In addition it has been shown that CYP2C9 has a very specific catalytic activity this carbon as well(2). The mechanism that the CYP's use is shown below

The P450 catalytic cycle in hydroxylation



The project:
It is our intention to insert a plasmid into E. Coli containing a promoter that we control as well as the sequence for one of the CYP's. After that we will turn our bacteria into cytochrome p450 factories in order to create enough so that they can convert a lot of 17beta-estradiol into 2hydroxyestradiol. As a courtesy of Quo Data we have been granted a A-Yes-Assay kit, that allows us to test how many compounds in water that reacts with the estrogen receptors. Hopefully if our CYP's are effective, we will observe that the ammount of compounds that react with the receptors will decrease.



Ecological and economical prospects:
Another 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.

References:
1. Schutze, N., Vollmer, G., Wunsche, W., Grote, A., Feit, B., and Knuppen, R. (1994) Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to the estrogen receptor of MCF-7 cells in cytosolic extracts and in nuclei of intact cells, Exp Clin Endocrinol 102, 399-408.
2. Lee, A. J., Cai, M. X., Thomas, P. E., Conney, A. H., and Zhu, B. T. (2003) Characterization of the oxidative metabolites of 17beta-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms, Endocrinology 144, 3382-3398.

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