Cytochrome P-450

Cytochrome P450
Cytochrome P450 Oxidase (CYP2C9)
Identifiers
Symbol p450
Pfam InterPro PROSITE PDOC00081
SCOP SUPERFAMILY OPM superfamily OPM protein 2bdm

The cytochrome P450 superfamily of monooxygenases (officially abbreviated as CYP) is a large and diverse group of enzymes that catalyze the oxidation of organic substances. The substrates of CYP enzymes include metabolic intermediates such as lipids and steroidal hormones, as well as xenobiotic substances such as drugs and other toxic chemicals. CYPs are the major enzymes involved in drug metabolism and bioactivation, accounting for about 75% of the total number of different metabolic reactions.[1]

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 (RH) while the other oxygen atom is reduced to water:

RH + O2 + NADPH + H+ → ROH + H2O + NADP+

Cytochromes P450 (CYPs) belong to the superfamily of proteins containing a heme cofactor and, therefore, are hemoproteins. CYPs use a variety of small and large molecules as substrates in enzymatic reactions.They 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 (450 nm) when it is in the reduced state and complexed with CO.

CYP enzymes have been identified in all domains of life - animals, plants, fungi, protists, bacteria, archaea, and even in viruses.[2] However, the enzymes have not been found in E. coli.[3][4] More than 18,000 distinct CYP proteins are known.[5]

Most CYPs require a protein partner to deliver one or more electrons to reduce the iron (and eventually molecular oxygen). Based on the nature of the electron transfer proteins CYPs can be classified into several groups:[6]

  • Microsomal P450 systems in which electrons are transferred from NADPH via cytochrome P450 reductase (variously CPR, POR, or CYPOR). Cytochrome b5 (cyb5) can also contribute reducing power to this system after being reduced by cytochrome b5 reductase (CYB5R).
  • Mitochondrial P450 systems, that employ adrenodoxin reductase and adrenodoxin to transfer electrons from NADPH to P450.
  • Bacterial P450 systems, that 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 sp. 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 CYP5 (thromboxane synthase), CYP8 (prostacyclin synthase), and CYP74A (allene oxide synthase).

Nomenclature

Genes encoding CYP enzymes, and the enzymes themselves, are designated with the abbreviation CYP, 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 (acetaminophen) metabolism. The CYP nomenclature is the official naming convention, although occasionally (and incorrectly) CYP450 or CYP450 is used. 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 (ThromBoXane A2 Synthase 1), and CYP51A1, lanosterol 14-α-demethylase, sometimes unofficially abbreviated to LDM according to its substrate (Lanosterol) and activity (DeMethylation).[7]

The current nomenclature guidelines suggest that members of new CYP families share >40% amino acid identity, while members of subfamilies must share >55% amino acid identity. There are nomenclature committees that assign and track both base CYP Allele Nomenclature Committee).

Mechanism

The active site of cytochrome P450 contains a heme iron center. The iron is tethered to the P450 protein via a thiolate ligand derived from a cysteine residue. This cysteine and several flanking residues are highly conserved in known CYPs and have the formal PROSITE signature consensus pattern [FW] - [SGNH] - x - [GD] - {F} - [RKHPT] - {P} - C - [LIVMFAP] - [GAD].[8] 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:

  1. The substrate binds to the active site of the enzyme, in close proximity to the heme group, on the side opposite to the peptide chain. The bound substrate 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,[9] and sometimes changing the state of the heme iron from low-spin to high-spin.[10] This gives rise to a change 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 spectrometry and is referred to as the "type I" difference spectrum (see inset graph in figure). 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 (see inset graph in figure). If no reducing equivalents are available, this complex may remain stable, allowing the degree of binding to be determined from absorbance measurements in vitro[11]
  2. The change in the electronic state of the active site favors the transfer of an electron from NAD(P)H via cytochrome P450 reductase or another associated reductase[12] This takes place by way of the electron transfer chain, as described above, reducing the ferric heme iron to the ferrous state.
  3. Molecular oxygen binds covalently to the distal axial coordination position of the heme iron. The cysteine ligand is a better electron donor than histidine, which is normally found in heme-containing proteins. As a consequence, the oxygen is activated to a greater extent than in other heme proteins. However, this sometimes allows the iron-oxygen bond to dissociate, causing the so-called "uncoupling reaction", which releases a reactive superoxide radical and interrupts the catalytic cycle.[9]
  4. A second electron is transferred via the electron-transport system, from either cytochrome P450 reductase, ferredoxins, or cytochrome b5, reducing the dioxygen adduct to a negatively charged peroxo group. This is a short-lived intermediate state.
  5. The peroxo group formed in step 4 is rapidly protonated twice by local transfer from water or from surrounding amino-acid side-chains, releasing one water molecule, and forming a highly reactive species commonly referred to as P450 Compound 1 ( or Compound I). This highly reactive intermediate was not "seen in action" until 2010,[13] although it had been studied theoretically for many years.[9] P450 Compound 1 is most likely an iron(IV)oxo (or ferryl) species with an additional oxidizing equivalent delocalized over the porphyrin and thiolate ligands. Evidence for the alternative perferryl iron(V)-oxo [9] is lacking.[13]
  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.

S: An alternative route for mono-oxygenation is via the "peroxide shunt": Interaction with single-oxygen donors such as peroxides and hypochlorites can lead directly to the formation of the iron-oxo intermediate, allowing the catalytic cycle to be completed without going through steps 2, 3, 4, and 5.[11] A hypothetical peroxide "XOOH" is shown in the diagram.

C: If carbon monoxide (CO) 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[14] 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 (or a very few) substrates, such as CYP19 (aromatase), 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 (including estrogen and testosterone synthesis and metabolism), 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.[15]

Drug metabolism


Further information: Drug metabolism

CYPs are the major enzymes involved in drug metabolism, accounting for about 75% of the total metabolism.[1] 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.

Drug interaction

Many drugs may increase or decrease the activity of various CYP isozymes either by inducing the biosynthesis of an isozyme (enzyme induction) or by directly inhibiting the activity of the CYP (enzyme inhibition). This is 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 take into account when using drugs of vital importance to the patient, drugs with important side-effects and drugs with small therapeutic windows, but any drug may be subject to an altered plasma concentration due to altered drug metabolism.

A classical example includes anti-epileptic drugs. Phenytoin, for example, induces CYP1A2, CYP2C9, CYP2C19, and CYP3A4. Substrates for the latter may be drugs with critical dosage, like amiodarone or carbamazepine, whose blood plasma concentration may either increase because of enzyme inhibition in the former, or decrease because of enzyme induction in the latter.

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.[17] Because of this risk, avoiding grapefruit juice and fresh grapefruits entirely while on drugs is usually advised.[18]

Other examples:

  • At relatively high concentrations, starfruit juice has also been shown to inhibit CYP2A6 and other CYPs.[22] Watercress is also a known inhibitor of the Cytochrome P450 CYP2E1, which may result in altered drug metabolism for individuals on certain medications (ex., chlorzoxazone).[23]

Other specific CYP functions

A subset of cytochrome P450 enzymes play important roles in the synthesis of steroid hormones (steroidogenesis) by the adrenals, gonads, and peripheral tissue:

CYP families in humans

Humans have 57 genes and more than 59 pseudogenes divided among 18 families of cytochrome P450 genes and 43 subfamilies.[24] 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.[15]

Family Function Members Names
CYP1 drug and steroid (especially estrogen) metabolism 3 subfamilies, 3 genes, 1 pseudogene CYP1A1, CYP1A2, CYP1B1
CYP2 drug and steroid metabolism 13 subfamilies, 16 genes, 16 pseudogenes CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1
CYP3 drug and steroid (including testosterone) metabolism 1 subfamily, 4 genes, 2 pseudogenes CYP3A4, CYP3A5, CYP3A7, CYP3A43
CYP4 arachidonic acid or fatty acid metabolism 6 subfamilies, 12 genes, 10 pseudogenes CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, CYP4X1, CYP4Z1
CYP5 thromboxane A2 synthase 1 subfamily, 1 gene CYP5A1
CYP7 bile acid biosynthesis 7-alpha hydroxylase of steroid nucleus 2 subfamilies, 2 genes CYP7A1, CYP7B1
CYP8 varied 2 subfamilies, 2 genes CYP8A1 (prostacyclin synthase), CYP8B1 (bile acid biosynthesis)
CYP11 steroid biosynthesis 2 subfamilies, 3 genes CYP11A1, CYP11B1, CYP11B2
CYP17 steroid biosynthesis, 17-alpha hydroxylase 1 subfamily, 1 gene CYP17A1
CYP19 steroid biosynthesis: aromatase synthesizes estrogen 1 subfamily, 1 gene CYP19A1
CYP20 unknown function 1 subfamily, 1 gene CYP20A1
CYP21 steroid biosynthesis 2 subfamilies, 1 gene, 1 pseudogene CYP21A2
CYP24 vitamin D degradation 1 subfamily, 1 gene CYP24A1
CYP26 retinoic acid hydroxylase 3 subfamilies, 3 genes CYP26A1, CYP26B1, CYP26C1
CYP27 varied 3 subfamilies, 3 genes CYP27A1 (bile acid biosynthesis), CYP27B1 (vitamin D3 1-alpha hydroxylase, activates vitamin D3), CYP27C1 (unknown function)
CYP39 7-alpha hydroxylation of 24-hydroxycholesterol 1 subfamily, 1 gene CYP39A1
CYP46 cholesterol 24-hydroxylase 1 subfamily, 1 gene CYP46A1
CYP51 cholesterol biosynthesis 1 subfamily, 1 gene, 3 pseudogenes CYP51A1 (lanosterol 14-alpha demethylase)

P450s in other species

Animals

Many animals have as many or more CYP genes than humans do. For example, mice have genes for 101 CYPs, and sea urchins have even more (perhaps as many as 120 genes).[25] Most CYP enzymes are presumed to have monooxygenase activity, as is the case for most mammalian CYPs that have been investigated (except for, e.g., CYP19 and CYP5). However, gene and genome sequencing is far outpacing biochemical characterization of enzymatic function, although many genes with close homology to CYPs with known function have been found.

The classes of CYPs most often investigated in non-human animals are those either involved in development (e.g., retinoic acid or hormone metabolism) or involved in the metabolism of toxic compounds (such as heterocyclic amines or polyaromatic hydrocarbons). Often there are differences in gene regulation or enzyme function of CYPs in related animals that explain observed differences in susceptibility to toxic compounds.

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 great model for cancer research in humans.[26] 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.[27]

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[28] and CYP6Z1 in the mosquito malaria vector Anopheles gambiae is capable of directly metabolizing DDT.[29]

Microbial

Microbial cytochromes P450 are often soluble enzymes and are involved in critical metabolic processes. Three examples that have contributed significantly to structural and mechanistic studies are listed here, but many different families exist.

  • Cytochrome P450cam (CYP101) originally from Pseudomonas putida has been used as a model for many cytochromes P450 and was the first cytochrome P450 three-dimensional protein structure solved by X-ray crystallography. This enzyme is part of a camphor-hydroxylating catalytic cycle consisting of two electron transfer steps from putidaredoxin, a 2Fe-2S cluster-containing protein cofactor.
  • Cytochrome P450 BM3 (CYP102A1) from the soil bacterium Bacillus megaterium catalyzes the NADPH-dependent hydroxylation of several long-chain fatty acids at the ω–1 through ω–3 positions. Unlike almost every other known CYP (except CYP505A1, cytochrome P450 foxy), it constitutes a natural fusion protein between the CYP domain and an electron donating cofactor. Thus, BM3 is potentially very useful in biotechnological applications.[30][31]
  • Cytochrome P450 119 (CYP119) isolated from the thermophillic archea Sulfolobus acidocaldarius [32] has been used in a variety of mechanistic studies.[13] Because thermophillic enzymes evolved to function at high temperatures, they tend to function more slowly at room temperature (if at all) and are therefore excellent mechanistic models.

Fungi

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. (This is useful only because humans' P450 have a different sensitivity; this is how this class of antifungals work.)[33]

Significant research is ongoing into fungal P450s, as a number of fungi are pathogenic to humans (such as Candida yeast and Aspergillus) and to plants.

Cunninghamella elegans is a candidate for use as a model for mammalian drug metabolism.

Plants

Plant cytochromes P450 are involved in a wide range of biosynthetic reactions, leading to various fatty acid conjugates, plant hormones, defensive compounds, or medically important drugs. Terpenoids, which represent the largest class of characterized natural plant compounds, are often substrates for plant CYPs.

P450s in biotechnology

The remarkable reactivity and substrate promiscuity of P450s have long attracted the attention of chemists.[34] Recent progress towards realizing the potential of using P450s towards difficult oxidations have included: (i) eliminating the need for natural co-factors by replacing them with inexpensive peroxide containing molecules,[35] (ii) exploring the compatibility of p450s with organic solvents,[36] and (iii) the use of small, non-chiral auxiliaries to predictably direct P450 oxidation.

InterPro subfamilies

InterPro subfamilies:

  • Cytochrome P450, B-class IPR002397
  • Cytochrome P450, mitochondrial IPR002399
  • Cytochrome P450, E-class, group I IPR002401
  • Cytochrome P450, E-class, group II IPR002402
  • Cytochrome P450, E-class, group IV IPR002403

References

External links

  • Performance of P450 inhibition Studies The performance of in vitro cytochrome P450 inhibition studies studies including analysis of the data.
  • DDI Regulatory Guidance Request a guide to drug-drug interaction regulatory recommendations.
  • Expanding the toolbox of cytochrome P450s through enzyme engineering Video by the Turner Group, University of Manchester, UK
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