cytochromes p450 · proteins. several cyp genes have already been identified, synthesized and...
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Introduction
Human impact on the environment presents an ongoing problem; with approximately 85,000
known chemicals being used today, we need to assure the safety of their use is assessed and
validated1. Various Cytochromes P450 (CYPs) are capable of metabolizing xenobiotic substances,
including potential environmental toxins. Selecting and expressing CYPs from model organisms that
are orthologous to the major drug metabolizing CYPs found in humans should allow for comparison
of their functions and assessment of their capacity in drug detoxification . Environmental
bio-indicator species (EBS) e.g. Danio rerio (Zebrafish) or Oncorhynchus mykiss (Rainbow Trout)
are of particular interest as they are often used as model species for monitoring the environment and
have similar biological pathways to other species2. By establishing the relationship between selected
environmental toxins and the products formed by action of EBS CYPs orthologous to human drug
metabolizing CYPs, I will ascertain the range of substrates, discover if the metabolites are similar to
their human counterparts, and establish if metabolites are potentially toxic and/or inhibit or induce
orthologue CYP activity.
Figure 1; Examples of important environmental toxins. These toxins are currently of concern to environmental agencies and they are known to be
found in the water table. These toxins were shown to be toxic to most aquatic organisms and also pose a threat to other species.
Cytochromes P450
CYPs are found in eukaryotes, numerous prokaryotes and also in the archaea. CYPs in the
human hepatic system have key roles in Phase I drug metabolism. They mediate diverse
(mostly oxidative) reactions on a large range of substrates. They are responsible for the
biotransformation of xenobiotics and ~75% of pharmaceuticals are metabolised by CYPs4.
During Phase I metabolism the biotransformation of the compound occurs, often
involving the addition of an oxygen atom (e.g. as a hydroxyl group) by a CYP enzyme,
resulting in a more hydrophilic product. However, CYP-dependent oxidative metabolism
can also result in other outcomes, including e.g. demethylation/dealkylation,
sulfoxidation, dehydration and epoxidation, as well as more “exotic” transformations
including isomerization, decarboxylation and C-C bond formation5. CYPs thus have
diverse catalytic activities and have been described as ‘Nature’s most versatile catalyst’5.
Environmentally Sensitive Species and their CYPs
Several species were selected as being of particular interest, since they are often used as model
organisms in research projects, and have also been used as bio-indicator species for environment
toxicology. These species play an important role in monitoring environmental pollution as they
demonstrate sensitivity to changes in aquatic environments. The use of biochemical markers such
as cytochromes P450 helps determine the mechanism of toxicity of a pollutant, providing insights
into the effects of the pollutant and its potential metabolites on the organism7. There is an
increasing emphasis on understanding the structure and function of key enzymes that are conserved
across species. Selecting key species that demonstrate similarity in relevant biological pathways to
other species (e.g. drug metabolism) can expedite the use of these non-traditional species as
toxicological models. In this project, I will examine species possessing orthologues of human CYP
genes, and establish if these orthologues may have conserved functions in metabolising xenobiotics
and/or can make non-human type metabolites that cause toxicity to environmentally sensitive
organisms8.
Conclusions and Ongoing Work
Cytochrome P450s have an important and evolving role in both pharmaceutical and
toxicological research. New and interesting data have shown how CYPs are involved in
adverse drug reactions, the toxicity of natural and man-made compounds and the activation
of toxins. How CYPs mediate these reactions is of great importance for a range of industries
and agencies. Microsomal CYPs are difficult to study but will provide a greater insight into
the mechanisms involved in xenobiotic transformation in our target species. To produce ESS
P450 enzymes, in vitro studies will be done using E. coli as the expression host. Selected
CYPs from the species discussed will be cloned into appropriate vectors to express the
proteins. Several CYP genes have already been identified, synthesized and cloned into
vectors. These CYP genes were synthesized to include a restriction site enabling (as required)
removal of the N-terminal transmembrane helix and were codon optimized for their host
E. coli (see Table 1). Preliminary results have revealed that five out of six initial clones show
CYP/CPR expression.
Cytochrome
p450 Species Class
Orthologous CYP
from Homo sapiens
% AA
Identity
CYP3A126 Pimephales promelas Teleost—Cyprinid CYP3A4 and CYP3A5 56.6/54.1
CYP3A27
Oncorhynchus mykiss
CYP3A4 and CYP3A5 53.3/52.3
Teleost—Salmonid
CYP3A45 52.9/52.7
CYP1A1
CYP1A2
51.6
CYP1A2 52.2
CYP1A3 52.4
CYP2K5 CYP2C9
44.2
CYP2M1 44.9
CPR CPR 72.4
CYP3A65
Danio rerio Teleost—Cyprinid
CYP3A4 and CYP3A5 54.1/51.8
CYP3C1-4 48.6/47.5
CYP1A CYP1A2 52.9
CPR CPR 73.9
Figure 4; The catalytic cycle of typical cytochromes P450. The stars indicate likely
rate-limiting steps in the activity of CYPs—which vary depending on the CYP. These include
the first electron reduction of P450 FeIII to FeII, and the dissociation of the product. The cy-
cle begins with substrate (RH) binding to displace the H2O ligand in the distal position)6.
Figure 3; Diagram showing the
heme binding pocket of CYP3A4
bound to ketoconazole. Ketocona-
zole is shown in turquoise, the heme is
shown in brick red and the secondary
protein structure are represented in
silver grey (PDB ID 2VoM). Three
molecules of ketoconazole are bound
in the CYP3A4 active site.
Figure 2. Diagram showing the
heme binding pocket of CYP3A4
bound to erythromycin. Erythro-
mycin is shown in turquoise, the heme
is shown in purple and the protein
secondary structure is represented in
green (PDB ID 2J0D)3.
Figure 5; Schematic of electron transfer reactions in microsomal and bacterial P450 redox systems. Panel A shows both a P450 and its cytochrome
P450 reductase (CPR) redox partner, both associated with the ER membrane through a N-terminal transmembrane anchor region. Electron transfer occurs from
NADPH through FAD and FMN cofactors to the P450 heme iron, ultimately leading to the activation of O2 bound to the heme iron and to its scission and the inser-
tion of an oxygen atom into the substrate (RH) to form a hydroxylated product (ROH). Panel B shows a similar reaction scheme for a soluble, bacterial P450-CPR
fusion enzyme (e.g. P450 BM3 [CYP102A1]). Neither of the domains of this enzyme have membrane anchor regions. (Adapted from Gueguen et al, 2006 )4.
Figure 6; Phylogenetic tree showing the relationship between CYPs in Homo sapiens and in three fish species of interest; The fish selected are
Pimephales promelas, Oncorhynchus mykiss and Danio rerio. This tree demonstrates the phylogenetic relationships between the species, and the orthologous rela-
tionships between the CYP enzymes. The functions of these CYPs may be largely conserved as the species have evolved and diverged.
Table 1; Orthologues identified by bioinformatics. Those genes shown in purple have been cloned and are undergoing expression trials.
References
1. Erikson, B. E., How many chemicals are in use today? EPA struggles to keep it's chemical inventory up
to date. Chem. Eng. News February 2017, 95 (9), 23-24
2. Siroka, Z.; Drastichova, J., Biochemical markers of aquatic environment contamination - Cytochrome
P450 in fish. A review. Acta Veterinaria Brno 2004, 73 (1), 123-132
3. Ekroos, M.; Sjogren, T., Structural basis for ligand promiscuity in cytochrome P450 3A4. Proc. Natl.
Acad. Sci. U. S. A. 2006, 103 (37), 13682-13687
4. Gueguen, Y.; Mouzat, K.; Ferrari, L.; Tissandie, E.; Lobaccaro, J. M. A.; Batt, A. M.; Paquet, F.; Voisin,
P.; Aigueperse, J.; Gourmelon, P.; Souidi, M., Cytochromes P450: xenobiotic metabolism, regulation
and clinical importance. Annales De Biologie Clinique 2006, 64 (6), 535-548
5. Coon, M. J., Cytochrome P450: nature's most versatile biological catalyst. Annu. Rev. Pharmacol. Tox-
icol. 2005, 45, 1-25
6. Corsini, A.; Bortolini, M., Drug-induced liver injury: the role of drug metabolism and transport. J.
Clin. Pharmacol. 2013, 53 (5), 463-474
7. Munro, A. W.; Girvan, H. M.; Mason, A. E.; Dunford, A. J.; McLean, K. J. What makes a P450 tick?
Trends Biochem. Sci. 2013, 38 (3), 140-150.
8. Siroka, Z.; Drastichova, J., Biochemical markers of aquatic environment contamination - Cytochrome
P450 in fish. A review. Acta Veterinaria Brno 2004, 73 (1), 123-132.
Emily Fox. Manchester Institute of Biotechnology, School of Chemistry, University of Manchester, 131 Princess Street, Manchester M1 7DN
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