Team:Copenhagen/Project/Cytochrome

Cytochrome P450s




What makes them special

Cytochromes p450 (called CYP) 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].

It's many functions have made it an appealing and interesting enzyme for synthetic biologist. Applications in different fields such as bioremidation, biosensors, drug develupment and cancer therapy have been explored - and cytochromes from plant to bacteria to human have all recieved attention. Standardization of Cytochromes as BioBricks will greatly benefit further research in all of these areas. [2]

We intend to submit 2 different types of CYP BioBricks to the partsregistry. One type is from the plant family CYP79 and the other type is from the mammalian cytochrome p450, family 1 and 2. We've chosen these to demonstrate the diverse applications for which cytochromes can be utilized.

The P450 catalytic cycle in hydroxylation

Cytochromes p450 are defined as heme-thiolate proteins featuring a particular spectral signature at 450 nm, thus the name [3]. 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 [4].

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 [5].

RH + NAD(P)H + O2 + H+ -> ROH + NAD(P)+ + H2O

This hydroxylation can yield many interesting compunds and is expensive to perform with ordinary methods. The substrates differ from CYP to CYP and whereas the plant CYP has high specificity for certain aminoacids which they converts to oximes, the human CYPs are more promiscuous and have very low specificity and modify many different substrates - of speciel interest are drugs, as human CYP are responsibly for 90% of the human bodys drug metabolism.

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 [5]. If CYPs are expressed in bacteria it is sometimes necesssary to supply the cell with CPR to provide it with enough NADPH - either coexpressing CYP and CPR or expressing them as a bicistron. [2] In our case, this is relevant for the human CYPs, and we intend to coexpress the cytochrome and reductase. The plant CYPs have previously shown to work fine in E.coli without the need for extra help.

References

[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
[2] Santosh KumarE: Engineering Cytochrome P450 Biocatalysts for Biotechnology, Medicine, and Bioremediation. Expert Opin Drug Metab Toxicol. 2010 February; 6(2): 115–131.
[3] Paul R. Ortiz de Montellano. Hydrocarbon Hydroxylation by Cytochrome P450 Enzymes. Chem Rev. 2010 February 10; 110(2): 932
[4] http://www.anaesthetist.com/physiol/basics/metabol/cyp/Findex.htm
[5] 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