Anna Radominska-Pandya Department of Biochemistry and Molecular Biology University of Arkansas for Medical Sciences Little Rock, Arkansas, US
October 2010; Gdansk University of Technology Outline
• Oxidoreductases • Cytochrome P450s (CYPs) – Superfamily – Structure – Mechanism – Phylogenetic Tree • Other reactions catalyzed by CYPs • Other oxidoreducatses and their reactions – Flavin Containing Monooxygenases (FMOs) Oxidoreductases (EC 1) Playing a Major or Secondary Role in Xenobiotic Metabolism
• Alcohol dehydrogenase (ADH; AKR1A1) • Aldehyde dehydrogenase (ALDH) • Aldehyde oxidase (AOX1) • Aldo-Keto reductases (AKR1) • Copper-containing amine oxidases (AOC) • CYTOCHROMES P450 (CYP) • Dihydrodiol dehydrogenases (DHDH; AKR1C) • Dopamine β-monooxygenase (DBH) • Flavin-containing monooxygenases (FMO) • Monoamine oxidases (MAO) • Myeloperoxidases (MPO) and other peroxidases • Prostaglandin-endoperoxide synthase (PTGS) • Quinone oxidoreductases (NQO) • Xanthine dehydrogenase/oxidase (XOR) Non-CYP Oxidoreductases
• Monoamine Oxidase (MAO) – MAO (mitochondrial) oxidatively deaminates endogenous substrates including neurotransmitters (dopamine, serotonin, norepinephrine, epinephrine); – Drugs designed to inhibit MAO used to affect balance of CNS neurotransmitters (L-DOPA) – MPTP converted to toxin MPP+ through MAO-B. • Diamine Oxidase (DAO) – DAO substrates include histamine and polyamines • Alcohol & Aldehyde Dehydrogenase – Non-specific enzymes found in soluble fraction of liver; ethanol metabolism • Xanthine Oxidase – Converts hypoxanthine to xanthine, and then to uric acid. Drug substrates include theophylline, 6-mercaptopurine. Allopurinol is substrate and inhibitor of xanthine oxidase; delays metabolism of other substrates; effective for treatment of gout. Non-CYP Oxidoreductases
• Flavin Monooxygenases – Family of enzymes that catalyze oxygenation of nitrogen, phosphorus, sulfur – particularly facile formation of N-oxides – Different FMO isoforms have been isolated from liver, lung (S.K. Krueger, et al. Drug Metab Rev 2002; 34:523-32) – Complete structures defined (Review: J. Cashman, 1995, Chem Res Toxicol 8:165-181; Pharmacogenomics 2002; 3:325-39) – Require molecular oxygen, NADPH, flavin adenosine dinucleotide (FAD) – Single point (loose) enzyme-substrate contact with reactive hydroperoxyflavin monoxoygenating agent – FMOs are heat labile and metal-free, unlike CYPs – Factors affecting FMOs (diet, drugs, sex) not as highly studied as CYPs The Cytochrome P450 Superfamily
CYPs The Cytochrome P450 (CYP) Superfamily
• Belong to the superfamily of proteins containing a heme cofactor and, therefore, are hemoproteins. • Named on the basis of their cellular (cyto) location and spectrophotometric characteristics (chrome): – When the reduced heme iron forms an adduct with CO, CYPs absorb light at wavelengths near 450 nm • CYP enzymes have been identified in all kingdoms of life – i.e., in animals, plants, fungi, bacteria, and archaea. • More than 11500 distinct CYP proteins The Cytochrome P450 (CYP) Superfamily
• Ancestral gene may date back circa 3.5 billion years • ~6,000 CYP genes are currently known – >60 in humans, excluding pseudogenes • CYP genes are present and expressed in all organisms except when lost through regressive mutations • Besides drugs, many other xenobiotics are also know CYP substrates – Sometimes with major toxicological consequences • Monooxygenation is by far their most frequent enzymatic mechanism – May also act as reductases, oxidases, and hydroperoxdases The Cytochrome P450 (CYP) Superfamily
• Predominant function is to catalyze the oxidation of organic substances • The substrates of CYP enzymes include: – Metabolic intermediates such as lipids and steroidal hormones – Xenobiotic substances such as drugs and other toxic chemicals • CYPs are the major enzymes involved in drug metabolism and bioactivation, accounting for ∼75% of the total metabolism. CYP Nomenclature
• Families - CYP plus arabic numeral – >40% homology of amino acid sequence, eg. CYP1 • Subfamily – 40-55% homology of amino acid sequence; eg. CYP1A – Additional arabic numeral when more than 1 subfamily has been identified; eg. CYP1A • Italics indicate gene (CYP1A2); regular font for enzyme CYP Families in Humans Humans have 57 genes and more than 59 pseudogenes divided among 18 families of cytochrome P450 genes and 43 subfamilies. Summary of CYP Genes and the Proteins They Encode.
Family Function Members Names drug and steroid metabolism 3 subfamilies, 3 genes CYP1 CYP1A1, CYP1A2, CYP1B1 (especially estrogen) 1 pseudogene CYP2A6, CYP2A7, CYP2A13, CYP2B6, 13 subfamilies, 16 genes CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2 drug and steroid metabolism 16 pseudogenes CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1 drug and steroid metabolism 1 subfamily, 4 genes, CYP3 CYP3A4, CYP3A5, CYP3A7, CYP3A43 (including testosterone) 2 pseudogenes CYP4A11, CYP4A22, CYP4B1, CYP4F2, arachidonic acid or fatty acid 6 subfamilies, 12 genes CYP4 CYP4F3, CYP4F8, CYP4F11, CYP4F12, metabolism 10 pseudogenes CYP4F22, CYP4V2, CYP4X1, CYP4Z1
CYP5 thromboxane A2 synthase 1 subfamily, 1 gene CYP5A1 bile acid biosynthesis 7-alpha CYP7 2 subfamilies, 2 genes CYP7A1, CYP7B1 hydroxylase of steroid nucleus CYP8A1 (prostacyclin synthase), CYP8 varied 2 subfamilies, 2 genes CYP8B1 (bile acid biosynthesis) CYP11 steroid biosynthesis 2 subfamilies, 3 genes CYP11A1, CYP11B1, CYP11B2 Summary of CYP Genes and the Proteins They Encode.
Family Function Members Names Steroid biosynthesis, CYP17 1 subfamily, 1 gene CYP17A1 17-alpha hydroxylase steroid biosynthesis: CYP19 1 subfamily, 1 gene CYP19A1 aromatase synthesizes estrogen CYP20 unknown function 1 subfamily, 1 gene CYP20A1 2 subfamilies, 1 gene CYP21 steroid biosynthesis CYP21A2 1 pseudogene CYP24 vitamin D degradation 1 subfamily, 1 gene CYP24A1
CYP26 retinoic acid hydroxylase 3 subfamilies, 3 genes CYP26A1, CYP26B1, CYP26C1 CYP27A1 (bile acid biosynthesis), CYP27 varied 3 subfamilies, 3 genes CYP27B1 (activates vitamin D3) 7-alpha hydroxylation of CYP39 1 subfamily, 1 gene CYP39A1 24-hydroxycholesterol CYP46 cholesterol 24-hydroxylase 1 subfamily, 1 gene CYP46A1 1 subfamily, 1 gene CYP51A1 (lanosterol 14-alpha CYP51 cholesterol biosynthesis 3 pseudogenes demethylase) CYP Structure: Hemoproteins
• CYPs are hemoproteins composed of: – A constant coenzyme (protoporphyrin IX) – A variable protein of ~50 kDa Surface of the Active Site of CYP2C9 Human Cytochrome P450 2A13
• Colored from: – blue N-terminus to red C-terminus. • Heme in gray. Membrane Location of CYPs
NADPH-CYP CYP Reductase (CPR) Heme 1e- + 1e-
Lipid bylayer of smooth endoplasmic reticulum Reactions Catalyzed by CYPs
• Reactions of monooxygenation – Oxidations of sp3-, sp2-C, and sp-C – N-oxidations – S-oxidation – O-oxidations – Oxidations of Si, P, As, etc. • Oxidase reaction -• – Liberation of superoxide (O2 ) • Hydroperoxidase reactions • Reactions of reduction Monooxygenase Reaction
• The most common reaction catalyzed by CYPs is a monooxygenase reaction, – e.g., insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water:
- + O2 + RH + 2e + 2H → ROH + H2O or - + O2 + R + 2e + 2H → RO + H2O
• Use a variety of small and large molecules as substrates in enzymatic reactions. • Often, they form part of multi-component electron transfer chains, called P450-containing systems. Monooxygenase Reaction
• NADPH + H+ + O2 + Drug → NADP+ + H2O + OH-Drug • Carbon monoxide binds to the reduced Fe(II) heme and absorbs at 450 nm (origin of enzyme family name) • Oxidative reactions require – CYP heme protein – CYP reductase – NADPH – Phosphatidylcholine – Molecular oxygen • CYPs are in smooth endoplasmic reticulum in close association with NADPH-CYP reductase in 10/1 ratio – The reductase serves as the electron source for the oxidative reaction cycle NADP+ Drug CYP Fe+3 CYP e- R-Ase Drug PC Drug OH NADPH
+3 CO CYP Fe CO CYP-Fe+2 CYP Fe+2 Drug u OH Drug h Drug e-
O2 +2 CYP Fe H2O O Drug 2 2H+
Electron flow in microsomal drug oxidizing system State of the Heme in CYP Catalytic cycle Spin States of CYPs
Low-spin form (s = 1/2) High-spin form (S = 5/2)
• Hexacoordinated • Pentacoordinated • Reduction potential approx. -360 to -300 mV • Reduction potential approx. -175 mV • Resting state (Y = hydroxy group or H2O) • Active (reducible) state • Inhibition (Y = xenobiotic hydroxy or amino)