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Cytochrome P450s in humans

David Nelson
Last modified Oct. 6, 2003


	Cytochrome P450 proteins in humans are drug metabolizing enzymes and 
enzymes that are used to make cholesterol, steroids and other important lipids 
such as prostacyclins and thromboxane A2.  These last two are metabolites of 
arachidonic acid.  Mutations in cytochrome P450 genes or deficiencies of the 
enzymes are responsible for several human diseases.  Induction of some P450s 
is a risk factor in several cancers since these enzymes can convert 
procarcinogens to carcinogens.  P450 enzymes play a major role in drug 
interactions.

	The name cytochrome P450 derives from the fact that these proteins 
have a heme group, and an unusual spectrum.  Mammalian cytochrome P450s are 
membrane bound.  They were originally discovered in rat liver microsomes.  
Microsomes are turbid suspensions made by grinding up cells and isolating the 
membrane fraction that is still in suspension after the cell debris and 
mitochondria have been pelleted.  These mixtures are very opaque to standard 
spectroscopy, because they scatter light so badly.  The only way to measure a 
spectrum on turbid samples like these was to make a special instrument with 
the light detector very close to the cuvette, and to use dual beams and do 
difference spectroscopy.  In this way all the interfering substances and the 
light scattering could be subtracted out.  With this setup, microsomes treated 
with dithionite (reduced microsomes) and with carbon monoxide gas added to one 
cuvette only give a very strong absorption band at 450 nm, thus P450 (P is for 
pigment).  This is called a reduced CO difference spectrum.  The CO binds tightly 
to the ferrous heme, giving a difference between the absorbance of the two 
cuvettes.  This spectrum was first observed in 1958.

	Other heme containing proteins don't absorb at 450 nm.  The reason why 
cytochrome P450 absorbs in this range is the unusual ligand to the heme iron.  
Four ligands are provided by nitrogens on the heme ring.  Above and below the 
plane of the heme, there is room for two more ligands, the 5th and 6th 
ligands.  In cytochrome P450s, the 5th ligand is a thiolate anion, a sulfur 
with a negative charge, S(-).  The sulfur comes from a conserved cysteine at 
the heme binding region of the active site.

	X-ray crystal structures are now available for more than ten different 
bacterial P450s (CYPs 51 [Mycobacterium], 55A1, 101A1, 102A1, 107A1 [eryF], 
111A1, 119A1, 121A1 [P450Mt2], 152, 175A1).
These proteins are soluble as compared to the eukaryotic P450s that are 
membrane bound.  The structures are similar and they probably form a 
good model for membrane bound versions, except for the membrane anchoring 
parts.  Here is a picture of the first P450 crystallized.  This is P450 cam 
from Pseudomonas putida, a bacterium that can use camphor as its sole carbon 
source.  This bug is found growing in soil under camphor trees.  The protein 
is shaped like a triangle with the heme buried deep inside.  In this 
structure, there is no access channel for substrate or products, even water, 
to enter or leave the active site.  Therefore, we must assume that the 
structure breathes when it functions, so a channel will be open at some point 
in the catalytic cycle.  Half of the enzyme is rich in alpha-helix and the 
other half is beta-sheet or non-repetetive structure.  The mammalian P450s are 
similar to this fold, but with an N-terminal membrane anchor.  A 
cartoon of one possible view is given here.  This model shows a single 
transmembrane segment, but membrane attachment is more complex than that.  
When the N-terminal sequence is removed the protein still sticks to membranes.  
The mammalian CYP2C5 protein has been crystallized after removal of the N-
terminal anchor peptide and replacement of an internal hydrophobic sequence 
with a more water soluble sequence from a related enzyme.  The X-ray structure 
has been solved (Williams PA, Cosme J, Sridhar V, Johnson EF, McRee DE. J Inorg 
Biochem 2000 Aug 31;81(3):183-90 Microsomal cytochrome P450 2C5: comparison to 
microbial P450s and unique features.) A brief description of the main 
features had appeared earlier (Arch. Biochem. Biophys. 369 Sept. 1, 24-29 1999).  The 
structure is similar to the soluble bacterial enzymes, but there are 
significant differences.

	The phamaceutical industry is very interested in P450 crystal structures with
drugs bound, so they can do improvement of drug design.  Here we see a press release 
from May 3, 2001 about a research agreement between Astex and AstraZeneca to determine 
crystal structures of human P450s with AstraZeneca drugs bound in the active site.  
Look at the outlined sections.  "Cytochrome P450 enzymes are the most prominent group 
of drug-metabolising enzymes in humans, and consequently are of great importance to the 
pharmaceutical industry.  ... Application of Astex’s technology to determine the three 
dimensional structures of human cytochrome P450 enxymes complexed with AstraZeneca’s 
compounds will facilitate rapid design of drug candidates with greater potential for 
clinical success.  AstraZeneca is one of the top five pharmaceutical companies in the 
world with 2000 healthcare sales of $15.8 billion."
Astex has now solved the crystal structures of CYP2C9 and CYP3A4, two of the most 
significant drug metabolising enzymes in humans.  This structure can now be used 
to modify existing drugs to make them poorer substrates for 3A4 (or better 
substrates).  Poorer P450 substrates would last longer in the body before 
elimination, which is desirable for the pharmaceutical industry.

	P450s catalyze many types of reactions, but the one that is most 
important for us is hydroxylation.  These enzymes are called mixed function 
oxidases or monooxygenases, because they incorporate one atom of molecular 
oxygen into the substrate and one atom into water.  They differ from 
dioxygenases that incorporate both atoms of molecular oxygen into the 
substrate.

	Foreign chemicals or drugs are also called xenobiotics.  Cytochrome 
P450s play an important role in xenobiotic metabolism, especially for 
lipophilic drugs.  The metabolism of these compounds takes place in two 
phases.  Phase I is chemical modification to add a functional group that can 
be used to attach a conjugate.  The conjugate makes the modified compound more 
water soluble so it can be excreted in the urine.  Many P450s add a hydroxyl 
group in a Phase I step of drug metabolism.  The hydroxyl then serves as the 
site for further modifications in Phase 2 drug metabolism.

	For cytochrome P450s to function, they also need a source of 
electrons.  The addition of two electrons (reduction) to the heme iron makes 
the difficult chemistry of breaking the oxygen-oxygen bond possible.  The 
electrons are donated by another protein that binds briefly to the P450 and 
passes an electron from a prosthetic group.  This handoff of electrons between 
proteins is called an electron transfer chain, and it is similar to the 
electron transfers that go on in complexes I to IV of the electron transfer 
chain in mitochondria.  (However, this is not the same electron transfer 
chain.)

	There are two different kinds of electron transfer chains for 
cytochrome P450s.  These depend on the location of the enzyme in the cell.  
Some P450s are found in the mitochondrial inner membrane and some are found in 
the endoplasmic reticulum (ER).  Both types of P450s are membrane bound proteins.  
The protein that donates electrons to P450s in the ER is called NADPH cytochrome 
P450 reductase.  It is also membrane bound by an N-terminal tail that crosses 
the ER membrane once.  The bulk of this protein is on the cytosolic side of 
the ER membrane.  This protein has two domains that each contain one flavin.  
Two electrons are acquired from NADPH and migrate from FAD to FMN, then to the 
P450 heme iron.

	In the mitochondria, the electron transfer chain is a little longer.  
Ferredoxin (called adrenodoxin in the adrenals, but exactly the same gene 
codes for both proteins) is the immediate donor of electrons to the P450s in 
mitochondria (CYP11A1, CYP11B1, CYP11B2, CYP24, CYP27A1, CYP27B1, CYP27C1).  
Ferredoxin has an iron sulfur cluster instead of a flavin, however, ferredoxin 
is reduced by ferredoxin reductase (or adrenodoxin reductase in the adrenals) 
that does contain a flavin.  NADPH is the source of electrons that flow from 
ferredoxin reductase to ferredoxin and then to P450.  A few P450s also can 
accept electrons from cytochrome b5.  This is a small membrane bound heme 
containing protein that gets its reducing equivalents (electrons) from NADH.


The families of human P450s


	The P450 proteins are categorized into families and subfamilies by 
their sequence similarities.  Sequences that are greater than 40% identical at 
the amino acid level belong to the same family.  Sequences that are greater 
than 55% identical are in the same subfamily.  There are now more than 2500 
cytochrome P450 sequences known.

Humans have 18 families of cytochrome P450 genes and 43 subfamilies

CYP1 drug metabolism (3 subfamilies, 3 genes, 1 pseudogene)
CYP2 drug and steroid metabolism (13 subfamilies, 16 genes, 16 pseudogenes)
CYP3 drug metabolism (1 subfamily, 4 genes, 2 pseudogenes)
CYP4 arachidonic acid or fatty acid metabolism (5 subfamilies, 11 genes, 10 
pseudogenes)
CYP5 Thromboxane A2 synthase (1 subfamily, 1 gene)
CYP7A bile acid biosynthesis 7-alpha hydroxylase of steroid nucleus (1 
subfamily member)
CYP7B brain specific form of 7-alpha hydroxylase (1 subfamily member)
CYP8A prostacyclin synthase (1 subfamily member)
CYP8B bile acid biosynthesis (1 subfamily member)
CYP11 steroid biosynthesis (2 subfamilies, 3 genes)
CYP17 steroid biosynthesis (1 subfamily, 1 gene) 17-alpha hydroxylase
CYP19 steroid biosynthesis (1 subfamily, 1 gene) aromatase forms estrogen
CYP20 Unknown function (1 subfamily, 1 gene)
CYP21 steroid biosynthesis (1 subfamily, 1 gene, 1 pseudogene)
CYP24 vitamin D degradation (1 subfamily, 1 gene)
CYP26A retinoic acid hydroxylase important in development (1 subfamily member)
CYP26B probable retinoic acid hydroxylase (1 subfamily member)
CYP26C probabvle retinoic acid hydroxylase (1 subfamily member)
CYP27A bile acid biosynthesis (1 subfamily member)
CYP27B Vitamin D3 1-alpha hydroxylase activates vitamin D3 (1 subfamily member)
CYP27C Unknown function (1 subfamily member)
CYP39 7 alpha hydroxylation of 24 hydroxy cholesterol (1 subfamily member)
CYP46 cholesterol 24-hydroxylase (1 subfamily member)
CYP51 cholesterol biosynthesis (1 subfamily, 1 gene, 3 pseudogenes) lanosterol 
14-alpha demethylase

Humans have 57 sequenced CYP genes and 58 pseudogenes.

only full length functional genes are named below

1A1, 1A2, 1B1, 2A6, 2A7, 2A13, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 
2J2, 2R1, 2S1, 2U1, 2W1, 3A4, 3A5, 3A7, 3A43, 4A11, 4A22, 4B1, 4F2, 4F3, 
4F8, 4F11, 4F12, 4F22, 4V2, 4X1, 4Z1 5A1, 7A1, 7B1, 8A1, 8B1, 11A1, 11B1, 
11B2, 17, 19, 20, 21A2, 24, 26A1, 26B1, 26C1, 27A1, 27B1, 27C1, 39, 46, 51, 

Detailed information on mouse, human, dog, cattle, rat and other species P450s
can be found on my website http://drnelson.utmem.edu/CytochromeP450.html 

A P450 name followed by P stands for a pseudogene.  A pseudogene is a defective 
gene that does not produce a functional protein.  There are several reasons 
why this happens, but in the end, the protein product is not made.  
Pseudogenes are relics of gene duplications where one of the copies has 
degenerated and lost its function.


Induction of P450 enzymes.


	P450 enzymes have a variety of gene regulatory mechanisms.  Many of 
these genes can be turned on or induced by a chemical signal.  The steroid 
hormones are under strict endocrine control.  Their levels are tightly 
regulated.  One example is the induction of steroid biosynthetic P450s by ACTH 
adrenocorticotropic hormone.  ACTH stimulates production of cAMP that 
presumably activates a protein kinase that phosphorylates some unidentified 
protein, leading to an increase in gene transcription.  

	Another type of P450 gene regulation is that shown by peroxisome 
proliferators like clofibrate.  These drugs act through a binding protein 
called the PPAR or peroxisome proliferator activated receptor.  When drug is 
bound to this protein it migrates to the nucleus, heterodimerizes with 
retinoid X receptor (RXR) and binds to specific DNA sequences in the 
regulatory region of genes that are needed for peroxisome generation.  The 
CYP4A1 gene is turned on by this mechanism. Peroxisomes oxidize fatty acids 
and the 4A1 P450 is a known fatty acid hydroxylase.

	The members of the CYP1 family are induced by aromatic hydrocarbons.  
The activation involves a specialized receptor called the Ah receptor.  Ah 
stands for aryl hydrocarbon.  This receptor protein binds the aromatic 
hydrocarbon, but it cannot reach the nucleus to activate gene transcription 
without another protein called arnt for Ah receptor nuclear translocator.  
These two proteins bind and together they then bind DNA and activate 
transcription.  

	Other chemicals also induce P450s. Ethanol induces the CYP2E enzymes.  
Phenobarbital induces the rat CYP2B enzymes 40-50 fold, through a 
phenobarbital receptor called CAR.  This receptor also dimerizes with RXR as 
seen above with the PPAR receptor.  The heterodimer binds to a phenobarbital 
response element in the DNA to activate the gene.  For details on these 
receptor mediated induction mechanisms see the review by Waxman 
(Archives Biochem. Biophys. 369, 11-23, 1999).  The general feature that many 
P450 enzymes are inducible is probably related to P450's role in 
detoxification of foreign chemicals found in plants.  


Noninvasive markers for measuring levels of P450 enzymes in humans


	P450 enzymes catalyze specific reactions that can be monitored by 
sampling the urine, blood or breath of patients given a noninvasive marker.  
Caffeine is a marker for CYP1A2.  It is demethylated, and the rate at which it 
is demethylated is related to the amount of CYP1A2 in a person's liver.  By 
administering caffeine and measuring the rate of demethylation, it is possible 
to estimate the level of CYP1A2 in a human.  This can show if a person has been 
induced by exposure to polycyclic aromatic hydrocarbons (PAHs). There are a 
variety of non-invasive markers for different P450s.  Assays of CYP1A enzymes 
from fish livers can also be used to monitor water pollution levels, since certain 
types of pollutants will induce the enzyme.  This is also being done in soil using 
nematodes like C. elegans.


Functions of human P450s and diseases caused by defects in P450s


	The CYP1 family of P450s can hydroxylate estrogen (CYP1A2 and 1B1)
and oxidize uroporphyrinogen to uroporphyrin (CYP1A2) in heme metabolism,
but they may have additional undiscovered endogenous substrates.  These 
enzymes are inducible by some polycyclic hydrocarbons, some of which are found 
in cigarette smoke and charred food.  These enzymes are of interest, because in 
assays, they can activate compounds to carcinogens.  High levels of CYP1A2 have 
been linked to an increased risk of colon cancer.  Since the 1A2 enzyme can be 
induced by cigarette smoking, this links smoking with colon cancer.  

	The CYP1B1 gene has been linked to primary congenital glaucoma
(See Human Molecular Genetics 6, 641-7, 1997; Am J Hum Genet. 62, 325-33 1998; 
Am J Hum Genet. 62, 573-84 1998; J Med Genet. 36, 290-4 1999).  The normal 
substrate in mammals is not known, but it is speculated that this P450 may be 
required to eliminate a signaling molecule.  Defects in the gene could lead to 
chronic high concentrations of the signaling molecule that lead to glaucoma.  
The molecule affected may be a steroid.

	As you can see from the table of human P450s, the 2 family is the 
largest family in humans.  About one third of human P450s are in this family.  
Many of these proteins can hydroxylate steroids, and some of them are 
expressed in a sex specific manner.  This would be expected for enzymes that 
only act on sex specific steroids.  Some of these may also be drug metabolism 
enzymes that are defensive, to protect us from toxins in our food.  Plants 
especially make many toxic components that are probably defensive for the 
plants.  Since we eat almost anything, it is necessary to have a 
detoxification system coded in our genes.  This idea has been called plant 
animal warfare on the chemical level.

	CYP2B is inducible by barbiturates in rodents.  It was one of 
the first P450s to be purified from mammals, but its role in humans is not 
understood. 

	CYP2C8 is known to catalyze the 6-alpha hydroxylation of taxol.  
This is a drug used in treating breast cancer.  

	CYP2C9 is one of two human P450s that has a known crystal structure. 
The other is CYP3A4 (still confidential).  CYP2C9 structure was published in 
Nature this summer (Williams PA, Cosme J, Ward A, Angove HC, Matak Vinkovic D, 
Jhoti H. Crystal structure of human cytochrome P450 2C9 with bound warfarin.
Nature. 2003 Jul 24;424, 464-468.)

	CYP2C19 metabolizes omeprazole, a common ulcer medication.
Polymorphisms in this gene cause a higher incidence of poor metabolizer phenotypes 
in Asians (23%) vs caucasians (3-5%).

    Drug metabolism differences caused by polymorphisms in P450s.

	A polymorphism is a difference in DNA sequence found at 1% or higher 
in a population.  These differences in DNA sequence can lead to differences in 
drug metabolism, so they are important features of P450 genes in humans.  
CYP2C19 has a polymorphism that changes the enzyme's ability to metabolize 
mephenytoin (a marker drug).  In Caucasians, the polymorphism for the poor 
metabolizer phenotype is only seen in 3% of the population.  However, it is 
seen in 20% of the asian population.  Because of this difference, it is 
important to be aware of a person's race when drugs are given that are 
metabolized differently by different populations.  Some drugs that have a 
narrow range of effective dose before they become toxic might be overdosed in 
a poor metabolizer.  Very recently, Roche has marketed a CYP450 DNA chip to detect 
major known polymorphisms in human CYP2D6 and CYP2C19.  For about $400 you can 
test a person to see if they are a poor metabolizer, normal metabolizer or ultra 
metabolizer, for a large number of drugs.  Since 1A2, 2C9, 2C19, 2D6 and 3A4 are 
responsible for oxidizing more than 90% of currently used drugs (2C9 paper above), 
this is a significant beginning to characterizing risk of adverse drug reactions 
in people. A cytochrome P450 allele website is available from Sweden 
at http://www.imm.ki.se/CYPalleles/

	CYP2D6 is perhaps the best studied P450 with a drug metabolism 
polymorphism.  This enzyme is responsible for more than 70 different drug 
oxidations.  Since there may be no other way to clear these drugs from the 
system, poor metabolizers may be at severe risk for adverse drug reactions.  
I heard a statistic at a meeting that adverse drug reactions are the number 4 
cause of hospitalization in the US.  There are at least 72 named alleles 
identified in CYP2D6.

CYP2D6 Substrates


oxycodone is oxycontin, a favorite drug of abuse.

	CYP2E1 is induced in alcoholics.  There is a polymorphism 
associated with this gene that is more common in Chinese people.  The mutation 
correlates with a 2-fold increased risk of nasopharyngeal cancer linked to 
smoking.  This is the second P450 enzyme that may be related to smoking 
induced cancer (see 1A2 above).  

	The CYP3A subfamily is one of the most important drug 
metabolizing families in humans.  The crystal structure of 3A4 is known, but 
confidential and heavily patented.  CYP3A4 is "the most abundantly expressed 
P450 in human liver". (Arch. Biochem. Biophys. 369, 11-23 1999)  The color of 
perfused liver is due to this protein.  CYP3A4 is known to metabolize more than 
120 different drugs.  Some of these are well known and I give a list here of some 
of the recognizable ones.

CYP3A4 Substrates


	Poisoning by acetominophen overdose is caused by P450 enzymes in the 
liver and kidney that convert acetominophen into a very toxic intermediate 
that can react with cellular macromolecules to damage cells and eventually 
kill them.  This intermediate normally reacts with glutathione, a natural 
antioxidant in cells.  It is only when the glutathione is depleted that cell 
death can occur.  That's why acetominophen overdoses don't have any serious 
symptoms until 3-4 days later.  This problem is worse in alcoholics, since 
they have induced P450 enzymes that make more of the toxic intermediate.

	There are common drugs given for special purposes that inhibit P450 
enzymes.  These include erythromycin (an antibiotic), ketoconazole, and 
itraconazole (both antifungals that inhibit the fungal CYP51 and unintentionally 
they also inhibit CYP3A4).  If these drugs are given with other drugs that are 
normally metabolized by P450 enzymes, the lifetime of these other drugs will be 
prolonged, and plasma levels will be increased, since they won't be cleared as fast.  
If these drugs affect heart rhythms or other critical systems, the result can 
be fatal.  For example, inhibition of CYP3A4 in a patient taking warfarin can 
cause bleeding.

	This is called a drug interaction.  Drug interactions are one of the major 
causes of death in hospitalized patients.  The risk of an adverse drug interaction 
increases with the number of drugs taken, with a probability of 40% when 10 drugs or 
more are taken.  The most seroius cases are due to drug metabolism by p450 enzymes.
A case report of a 63 year old man receiving medication for major depression showed 
he boarded a plane in Toronto to fly to London.  On arrival he was unrousable.  In his 
Carry-on bag he had Mefadazone (for depression), Ketoconazole (for fungal infection)
and Triazolam (an antipsychotic also used for insomnia).  All three of these drugs 
bind to CYP3A4.  Ketoconazole inhibits CYP3A4 and probably caused the other two 
drugs to become overdosed.  

	Another example is terfenadine (a non-sedating anti-histamine) with ketoconazole.  
Studies in 1993 (Honig) showed a 15-72 fold increase in terfenadine AUC (Area under the 
curve) due to inhibition of CYP3A4 by ketoconazole. Torsades de pointes (TDP) is a 
potentially fatal ventricular tachycardia. TDP is a side-effect that has led to withdrawal 
of several drugs from the market including terfenadine.  This is a case where a 72 fold 
increase in drug dose might harm or even kill a patient.  

	Another factor in drug dosage is interfering substances from food.  Grapefruit 
juice contains a CYP3A4 inhibitor that causes about a 12 fold increase in some drug 
concentrations.  And the effect lasts for several days.  It is advisable to discourage 
your patients from drinking grapefruit juice while on medication metabolized by CYP3A4.

	Now we will leave the drug metabolizing enzymes behind and talk about 
P450s that are very specific in their reactions, just the opposite of CYP3A4.  
These enzymes tend to be in families with one or two members and they have 
only one substrate.  Most of these enzymes use steroids or steroid precursors 
as their substrates.  

	CYP5 is the thromboxane A2 synthase.  Thromboxane A2 is a fatty 
acid in the arachidonic acid cascade.  Arachidonic acid can be metabolized in 
two pathways, the linear pathway that leads to leukotrienes, and the cyclic 
pathway that leads to prostaglandins and thromboxanes.  The first enzymes 
leading to cyclic products of arachidonic acid are cyclooxygenases 1 and 2.  
These enzymes are inhibited by aspirin and non-steroidal antiinflammatory drugs 
(NSAIDS).  Aspirin acetylates a serine in the enzyme that blocks the binding 
of arachidonic acid.  Current research shows that COX2 is inducible and is 
found to be induced in inflammation.  COX1 is constitutive.  This difference 
suggests that COX2 specific inhibitors would block inflammation while not 
interfering with the beneficial effects of COX1, such as maintaining the 
stomach lining.  These drugs are now on the market.  Mouse 
knockouts have been made, but the full analysis of these COX1 and COX2 
knockouts is not finished yet.  After this step the pathway branches.  Two of 
the branches include cytochrome P450 reactions.  One leads to thromboxane A2 
(CYP5) and the other to prostacyclin (CYP8A1).  Thromboxane A2 causes platelet 
aggregation and that is why aspirin prevents platelet aggregation.  
Prostacyclin acts in opposition to thromboxane A2.  It is a vasodilator and an 
inhibitor of platelet aggregation.  The acetylation of COX1 and COX2 in 
platelets is critical since the platelets have no nucleus and cannot 
resynthesize the inhibited enzymes.

	CYP7A is the first and rate limiting step of bile acid 
synthesis.  This pathway is the only means the body has of eliminating 
cholesterol in liver.  CYP39 can substitute for it in brain. As we will see later, 
CYP51 is a key enzyme in cholesterol biosynthesis, so P450s are active at both 
ends of cholesterol metabolism.  In 2002 patients were found with defects 
in this gene.  They had elevated levels of cholesterol, decreased levels of bile 
acids and increased triglycerides, as a compensation for the reduced bile acids. 
John Kane et al. Journal of Clin. Invest. July 2002.

	CYP7B  a novel brain cytochrome P450, catalyzes the synthesis 
of neurosteroids 7-alpha hydroxy dehydroepiandrosterone and 7-alpha hydroxy 
pregnenolone Proc. Natl. Acad. Sci. USA  94, 4925-4930 (1997)

	CYP8A is prostacyclin synthase (prostaglandin I2).  It is part 
of a regulatory component of hemostasis that opposes CYP5 that makes 
thromboxane A2.  

	CYP8B is the 12-alpha hydroxylase needed in bile acid 
biosynthesis

	CYP11A1 is the side chain cleavage enzyme that converts 
cholesterol to pregnenolone.  This is the first step in steroid biosynthesis.  
Defects in this enzyme lead to a lack of glucocorticoids, feminization and 
hypertension.  [mitochondrial]

	CYP11B1 is the 11-beta hydroxylase enzyme that can act on 11-
deoxycortisol to make cortisol or it can hydroxylate 11-deoxycorticosterone to 
make corticosterone.  [mitochondrial]  Defects in this gene lead to congenital 
adrenal hyperplasia.

	CYP11B2 is aldosterone synthase that hydroxylates coricosterone 
at the 18 position.  [mitochondrial]  Defects in this gene lead to congenital 
hypoaldosteronism.

	CYP17 is the 17 alpha hydroxylase and 17-20 lyase (two enzymes 
in one).  A mutation in this gene is described in Nature Genetics 17, 201-205 
(1997) that causes the loss of the 17-20 lyase activity without affecting the 
17 hydroxylase activity.  This enzyme is required for production of 
testosterone and estrogen.  Defects in this enzyme affect proper development 
at puberty.

	CYP19 is aromatase that makes estrogen by aromatizing the A 
ring of the steroid nucleus.  Lack of this enzyme causes a lack of estrogen 
and failure of women to develop at puberty.  An interesting defect found in a 
male was an overactive CYP19 enzyme with about 50 times normal activity.  This 
boy developed breasts at a young age.

	CYP20 is a new P450 found only on chordates so far, it may be 
chordate specific and be involved in development.  Nothing is known yet.

	CYP21 is the C21 steroid hydroxylase.  Defects in this gene 
cause congential adrenal hyperplasia due to lack of cortisol synthesis.  Since 
cortisol is not made, the precursor 17 hydroxy progesterone builds up and this 
causes excessive androgen (testosterone) biosynthesis resulting in 
virilization.  

	CYP24 is a 25-hydroxyvitamin D(3) 24-hydroxylase used in the 
degradation or inactivation of vitamin D metabolites.  [mitochondrial]

	CYP26A1 is an all trans retinoic acid hydroxylase.  It does not 
recognize 9-cis or 13-cis retinoic acid.  CYP26A1 has been mutated in 
zebrafish and it causes a developmental defect.  The human and mouse cDNAs 
have been cloned, but the effects of a mutation in mammals is not yet 
determined.  Retinoic acid is known to be an important molecule in vertebrate 
development.  It operates through several retinoic acid receptors.  The 
hydroxylase may be a means of degrading the retinoic acid signal and thus 
turning off a developmental switch.

	CYP26B1 is a recently discovered human P450. It metabolizes retinoic 
acid and its expression is induced by retinoic acid during development in chickens 
(and probably all vertebrates). 
(See Nelson, D.R. A second CYP26 P450 in humans and zebrafish: CYP26B1. 
Archives of Biochem. Biophys. 371, 345-347 1999 and
Gene Expr Patterns. 2003 Oct;3(5):621-7. 
Expression of the retinoic acid catabolising enzyme CYP26B1 in the chick embryo 
and its regulation by retinoic acid.
Reijntjes S, Gale E, Maden M.)

	CYP26C1 is only known from genomic DNA sequencing.  The 
function is not known.

	CYP27A1 is a sterol 27-hydroxylase that catalyzes the first 
step in side chain oxidation of sterol intermediates in bile acid 
biosynthesis. The sterol storage disorder cerebrotendinous xanthomatosis (CTX) 
is characterized by abnormal deposition of cholesterol and cholestanol in 
tissues like the Achilles tendon and nervous tissues.  This disease is caused 
by mutations in the CYP27A1 gene.  Remember that formation of bile acids is 
the only way the body can eliminate cholesterol, so if this pathway becomes 
blocked, then cholestrol can build up and become a problem.  The end products 
of bile acid synthesis are cholic acid and chenodeoxycholic acid.  These are 
the feedback inhibitors that shut down the biosynthesis of bile acids.  This 
disease can be treated by giving cholic acid to shut down the bile acid 
pathway.  CYP7A and CYP8B are two other enzymes in this bile acid biosynthesis 
pathway.  CYP27A1 also 25 hydroxylates vitamin D3. 

	CYP27B1 is the 1-alpha hydroxylase of vitamin D3 that converts 
the D3 precursor to the active vitamin form.  This gene was cloned after much 
effort, because the product acts to feedback inhibit mRNA systhesis.  The 
paper appeared in Science (Sept. 19, 1997) .  Because of this mechanism, it 
was very hard to get enough mRNA to clone this cDNA.  The trick that was used 
was to make a knockout mouse that was missing the vitamin D3 receptor.  This 
prevented the feedback inhibition and allowed a buildup of mRNA for the gene.  
[mitochondrial]

	CYP27C1 is only known from genomic DNA sequencing.  The 
function is not known. This gene appears to be missing in rodents, so it cannot be 
essential for vertebrate develpoment.

	CYP39 is the 7 hydroxylase of 24 hydroxy cholesterol

	CYP46 is the cholesterol 24 hydroxylase

	CYP51 is the lanosterol 14-alpha demethylase that is key in 
making cholestrol from lanosterol.  This is the target of the triazole 
antifungal drugs like ketoconazole.  This enzyme is evolutionarily conserved 
in plants, fungi and animals, and bacteria.  It is found in Mycobacterium 
tuberculosis.  This is the only P450 to be so highly conserved and it may have 
been the ancestor to all eukaryotic P450s.


Differences in humans, mice and rats.


	Not all mammals have the same exact sets of P450 enzymes.  They do 
tend to have the very specific ones we talked about for making steroids and 
bile acids, but they do not always have the same xenobiotic metabolizing 
P450s.  The 2D subfamily is an interesting example.  In humans there is only 
one active 2D P450, the 2D6 enzyme.  The 2D6 enzyme in humans is also the 
enzyme responsible for the debrisoquine hydroxylase polymorphism we talked 
about earlier.  In mice there are nine different functional Cyp2d P450 enzymes.  
Humans have one CYP2J2 while mice have eight Cyp2j P450s.  Humans have 4 CYP2cs
While mice have 15. This has implications for drug testing in animals.  One has 
to be concerned that studying the effect of a drug in mice or rats may not be 
relevant to humans, since the drug metabolizing systems are different.  Beagle 
dogs are sometimes used in drug experiments, because their drug metabolism is 
supposed to be closer to humans than rodents.  For a more detailed discussion 
see Nelson, D.R. Cytochrome P450 and the individuality of Species.  Archives of 
Biochem. Biophys. 369, Sept. 1 issue 1-10, 1999.


Use of a P450 for gene therapy in cancer


	I mentioned earlier that CYP1A2 can activate procarcinogens to 
carcinogens.  The induction of this enzyme may be a cancer risk.  The 
activation of a prodrug to an active form by a P450 mediated reaction has been 
exploited to fight cancer. A vector with a P450 gene on it (and a P450 
reductase gene) can be injected into cancer tumors.  Some of these cells take 
up the vector and express The P450 and its reductase.  Then a non-toxic 
prodrug is administered that is converted by the P450 into a toxic compound 
that kills the cells.  Since the cancer cells have cellular connections, the 
toxin gets shared around and the tumor dies. For a review on this approach to 
cancer therapy see Waxman DJ, Chen L, Hecht JE, Jounaidi Y Cytochrome P450-
based cancer gene therapy: recent advances and future prospects. Drug Metab 
Rev 31,503-22 1999.