Cytochrome P450


Cytochromes P450 are a superfamily of enzymes containing heme as a cofactor that function as monooxygenases. In mammals, these proteins oxidize steroids, fatty acids, and xenobiotics, and are important for the clearance of various compounds, as well as for hormone synthesis and breakdown. In plants, these proteins are important for the biosynthesis of defensive compounds, fatty acids, and hormones.
CYP enzymes have been identified in all kingdoms of life: animals, plants, fungi, protists, bacteria, and archaea, as well as in viruses. However, they are not omnipresent; for example, they have not been found in Escherichia coli. More than 50,000 distinct CYP proteins are known.
CYPs are, in general, the terminal oxidase enzymes in electron transfer chains, broadly categorized as P450-containing systems. The term "P450" is derived from the spectrophotometric peak at the wavelength of the absorption maximum of the enzyme when it is in the reduced state and complexed with carbon monoxide. Most CYPs require a protein partner to deliver one or more electrons to reduce the iron.

Nomenclature

s encoding CYP enzymes, and the enzymes themselves, are designated with the root symbol CYP for the superfamily, followed by a number indicating the gene family, a capital letter indicating the subfamily, and another numeral for the individual gene. The convention is to italicise the name when referring to the gene. For example, CYP2E1 is the gene that encodes the enzyme CYP2E1—one of the enzymes involved in paracetamol metabolism. The CYP nomenclature is the official naming convention, although occasionally CYP450 or CYP450 is used synonymously. However, some gene or enzyme names for CYPs may differ from this nomenclature, denoting the catalytic activity and the name of the compound used as substrate. Examples include CYP5A1, thromboxane A2 synthase, abbreviated to TBXAS1, and CYP51A1, lanosterol 14-α-demethylase, sometimes unofficially abbreviated to LDM according to its substrate and activity.
The current nomenclature guidelines suggest that members of new CYP families share at least 40% amino acid identity, while members of subfamilies must share at least 55% amino acid identity. There are nomenclature committees that assign and track both base gene names and allele names.

Classification

Based on the nature of the electron transfer proteins, CYPs can be classified into several groups:
;Microsomal P450 systems: in which electrons are transferred from NADPH via cytochrome P450 reductase. Cytochrome b5 can also contribute reducing power to this system after being reduced by cytochrome b5 reductase.
;Mitochondrial P450 systems: which employ adrenodoxin reductase and adrenodoxin to transfer electrons from NADPH to P450.
;Bacterial P450 systems: which employ a ferredoxin reductase and a ferredoxin to transfer electrons to P450.
;CYB5R/cyb5/P450 systems', in which both electrons required by the CYP come from cytochrome b5.
;FMN/Fd/P450 systems: originally found in Rhodococcus'' species, in which a FMN-domain-containing reductase is fused to the CYP.
;P450 only: systems, which do not require external reducing power. Notable ones include thromboxane synthase, prostacyclin synthase, and CYP74A.
The most common reaction catalyzed by cytochromes P450 is a monooxygenase reaction, e.g., insertion of one atom of oxygen into the aliphatic position of an organic substrate while the other oxygen atom is reduced to water:

RH + O2 + NADPH + H+ → ROH + H2O + NADP+
Many hydroxylation reactions use CYP enzymes.

Mechanism

Structure

The active site of cytochrome P450 contains a heme-iron center. The iron is tethered to the protein via a cysteine thiolate ligand. This cysteine and several flanking residues are highly conserved in known CYPs and have the formal PROSITE signature consensus pattern - - x - - - - - C - - . Because of the vast variety of reactions catalyzed by CYPs, the activities and properties of the many CYPs differ in many aspects. In general, the P450 catalytic cycle proceeds as follows:

Catalytic cycle

  1. Substrate binds in proximity to the heme group, on the side opposite to the axial thiolate. Substrate binding induces a change in the conformation of the active site, often displacing a water molecule from the distal axial coordination position of the heme iron, and changing the state of the heme iron from low-spin to high-spin.
  2. Substrate binding induces electron transfer from NADH via cytochrome P450 reductase or another associated reductase.
  3. Molecular oxygen binds to the resulting ferrous heme center at the distal axial coordination position, initially giving a dioxygen adduct not unlike oxy-myoglobin.
  4. A second electron is transferred, from either cytochrome P450 reductase, ferredoxins, or cytochrome b5, reducing the Fe-O2 adduct to give a short-lived peroxo state.
  5. The peroxo group formed in step 4 is rapidly protonated twice, releasing one molecule of water and forming the highly reactive species referred to as P450 Compound 1. This highly reactive intermediate was isolated in 2010, P450 Compound 1 is an iron oxo species with an additional oxidizing equivalent delocalized over the porphyrin and thiolate ligands. Evidence for the alternative perferryl iron-oxo is lacking.
  6. Depending on the substrate and enzyme involved, P450 enzymes can catalyze any of a wide variety of reactions. A hypothetical hydroxylation is shown in this illustration. After the product has been released from the active site, the enzyme returns to its original state, with a water molecule returning to occupy the distal coordination position of the iron nucleus.
utilized by cytochrome P450 for conversion of hydrocarbons to alcohols via the action of "compound I", an iron oxide bound to a heme radical cation.|300px
  1. An alternative route for mono-oxygenation is via the "peroxide shunt". This pathway entails oxidation of the ferric-substrate complex with oxygen-atom donors such as peroxides and hypochlorites. A hypothetical peroxide "XOOH" is shown in the diagram.

    Spectroscopy

Binding of substrate is reflected in the spectral properties of the enzyme, with an increase in absorbance at 390 nm and a decrease at 420 nm. This can be measured by difference spectroscopies and is referred to as the "type I" difference spectrum. Some substrates cause an opposite change in spectral properties, a "reverse type I" spectrum, by processes that are as yet unclear. Inhibitors and certain substrates that bind directly to the heme iron give rise to the type II difference spectrum, with a maximum at 430 nm and a minimum at 390 nm. If no reducing equivalents are available, this complex may remain stable, allowing the degree of binding to be determined from absorbance measurements in vitro
C: If carbon monoxide binds to reduced P450, the catalytic cycle is interrupted. This reaction yields the classic CO difference spectrum with a maximum at 450 nm.

P450s in humans

Human CYPs are primarily membrane-associated proteins located either in the inner membrane of mitochondria or in the endoplasmic reticulum of cells. CYPs metabolize thousands of endogenous and exogenous chemicals. Some CYPs metabolize only one substrates, such as CYP19, while others may metabolize multiple substrates. Both of these characteristics account for their central importance in medicine. Cytochrome P450 enzymes are present in most tissues of the body, and play important roles in hormone synthesis and breakdown, cholesterol synthesis, and vitamin D metabolism. Cytochrome P450 enzymes also function to metabolize potentially toxic compounds, including drugs and products of endogenous metabolism such as bilirubin, principally in the liver.
The Human Genome Project has identified 57 human genes coding for the various cytochrome P450 enzymes.

Drug metabolism

CYPs are the major enzymes involved in drug metabolism, accounting for about 75% of the total metabolism. Most drugs undergo deactivation by CYPs, either directly or by facilitated excretion from the body. Also, many substances are bioactivated by CYPs to form their active compounds like the antiplatelet drug clopidogrel.

Drug interaction

Many drugs may increase or decrease the activity of various CYP isozymes either by inducing the biosynthesis of an isozyme or by directly inhibiting the activity of the CYP. A classical example includes anti-epileptic drugs, such as Phenytoin, which induces CYP1A2, CYP2C9, CYP2C19, and CYP3A4.
Effects on CYP isozyme activity are a major source of adverse drug interactions, since changes in CYP enzyme activity may affect the metabolism and clearance of various drugs. For example, if one drug inhibits the CYP-mediated metabolism of another drug, the second drug may accumulate within the body to toxic levels. Hence, these drug interactions may necessitate dosage adjustments or choosing drugs that do not interact with the CYP system. Such drug interactions are especially important to consider when using drugs of vital importance to the patient, drugs with significant side-effects, or drugs with a narrow therapeutic index, but any drug may be subject to an altered plasma concentration due to altered drug metabolism.
Many substrates for CYP3A4 are drugs with a narrow therapeutic index, such as amiodarone or carbamazepine. Because these drugs are metabolized by CYP3A4, the mean plasma levels of these drugs may increase because of enzyme inhibition or decrease because of enzyme induction.

Interaction of other substances

Naturally occurring compounds may also induce or inhibit CYP activity. For example, bioactive compounds found in grapefruit juice and some other fruit juices, including bergamottin, dihydroxybergamottin, and paradicin-A, have been found to inhibit CYP3A4-mediated metabolism of certain medications, leading to increased bioavailability and, thus, the strong possibility of overdosing. Because of this risk, avoiding grapefruit juice and fresh grapefruits entirely while on drugs is usually advised.
Other examples:

Steroid hormones

A subset of cytochrome P450 enzymes play important roles in the synthesis of steroid hormones by the adrenals, gonads, and peripheral tissue:
Certain cytochrome P450 enzymes are critical in metabolizing polyunsaturated fatty acids to biologically active, intercellular cell signaling molecules and/or metabolize biologically active metabolites of the PUFA to less active or inactive products. These CYPs possess cytochrome P450 omega hydroxylase and/or epoxygenase enzyme activity.
Humans have 57 genes and more than 59 pseudogenes divided among 18 families of cytochrome P450 genes and 43 subfamilies. This is a summary of the genes and of the proteins they encode. See the homepage of the cytochrome P450 Nomenclature Committee for detailed information.

P450s in other species

Animals

Many animals have as many or more CYP genes than humans do. Reported numbers range from 35 genes in the sponge Amphimedon queenslandica to 235 genes in the cephalochordate Branchiostoma floridae. Mice have genes for 101 CYPs, and sea urchins have even more.
Most CYP enzymes are presumed to have monooxygenase activity, as is the case for most mammalian CYPs that have been investigated. Gene and genome sequencing is far outpacing biochemical characterization of enzymatic function, though many genes with close homology to CYPs with known function have been found, giving clues to their functionality.
The classes of CYPs most often investigated in non-human animals are those either involved in development or involved in the metabolism of toxic compounds. Often there are differences in gene regulation or enzyme function of CYPs in related animals that explain observed differences in susceptibility to toxic compounds. Some drugs undergo metabolism in both species via different enzymes, resulting in different metabolites, while other drugs are metabolized in one species but excreted unchanged in another species. For this reason, one species's reaction to a substance is not a reliable indication of the substance's effects in humans. A species of Sonoran Desert Drosophila that uses an upregulated expression of the CYP28A1 gene for detoxification of cacti rot is Drosophila mettleri. Flies of this species have adapted an upregulation of this gene due to exposure of high levels of alkaloids in host plants.
CYPs have been extensively examined in mice, rats, dogs, and less so in zebrafish, in order to facilitate use of these model organisms in drug discovery and toxicology. Recently CYPs have also been discovered in avian species, in particular turkeys, that may turn out to be a useful model for cancer research in humans. CYP1A5 and CYP3A37 in turkeys were found to be very similar to the human CYP1A2 and CYP3A4 respectively, in terms of their kinetic properties as well as in the metabolism of aflatoxin B1.
CYPs have also been heavily studied in insects, often to understand pesticide resistance. For example, CYP6G1 is linked to insecticide resistance in DDT-resistant Drosophila melanogaster and CYP6M2 in the mosquito malaria vector Anopheles gambiae is capable of directly metabolizing pyrethroids.

Microbial

Microbial cytochromes P450 are often soluble enzymes and are involved in diverse metabolic processes. In bacteria the distribution of P450s is very variable with many bacteria having no identified P450s. Some bacteria, predominantly actinomycetes, have numerous P450s. Those so far identified are generally involved in either biotransformation of xenobiotic compounds or are part of specialised metabolite biosynthetic pathways. Although no P450 has yet been shown to be essential in a microbe, the CYP105 family is highly conserved with a representative in every streptomycete genome sequenced so far. Due to the solubility of bacterial P450 enzymes, they are generally regarded as easier to work with than the predominantly membrane bound eukaryotic P450s. This, combined with the remarkable chemistry they catalyse, has led to many studies using the heterologously expressed proteins in vitro. Few studies have investigated what P450s do in vivo, what the natural substrate are and how P450s contribute to survival of the bacteria in the natural environment.Three examples that have contributed significantly to structural and mechanistic studies are listed here, but many different families exist.
The commonly used azole class antifungal drugs work by inhibition of the fungal cytochrome P450 14α-demethylase. This interrupts the conversion of lanosterol to ergosterol, a component of the fungal cell membrane.
Significant research is ongoing into fungal P450s, as a number of fungi are pathogenic to humans and to plants.
Cunninghamella elegans is a candidate for use as a model for mammalian drug metabolism.

Plants

Plant cytochrome P450s are involved in a wide range of biosynthetic reactions and target a diverse range of biomolecules. These reactions lead to various fatty acid conjugates, plant hormones, secondary metabolites, lignins, and a variety of defensive compounds. Plant genome annotations suggest that cytochrome P450 genes make up as much as 1% of the plant genes. The number and diversity of P450 genes is responsible, in part, for the multitude of bioactive compounds.
Cytochrome P450 aromatic O-demethylase, which is made of two distinct promiscuous parts: a cytochrome P450 protein and three domain reductase, is significant for its ability to convert Lignin, the aromatic biopolymer common in plant cell walls, into renewable carbon chains in a catabolic set of reactions. In short, it is a facilitator of a critical step in Lignin conversion.

P450s in biotechnology

The remarkable reactivity and substrate promiscuity of P450s have long attracted the attention of chemists. Recent progress towards realizing the potential of using P450s towards difficult oxidations have included: eliminating the need for natural co-factors by replacing them with inexpensive peroxide containing molecules, exploring the compatibility of P450s with organic solvents, and the use of small, non-chiral auxiliaries to predictably direct P450 oxidation.

InterPro subfamilies

subfamilies:
Clozapine, imipramine, paracetamol, phenacetin Heterocyclic aryl amines
Inducible and CYP1A2 5-10% deficient
oxidize uroporphyrinogen to uroporphyrin in heme metabolism, but they may have additional undiscovered endogenous substrates.
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.