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Home , CYP17A1, CYP20A1, CYP26A1, CYP26B1, CYP26C1, CYP27C1

Cytochrome P450: Oxygen activation and biodiversty

1 Biodiversity of P-450 :

Cross-talk between chemistry and biology

Heme Fe(II)-CO complex 450 nm, different from those of hemoglobin and other 410-420 nm. Pigment of 450 nm CYP3A4….

2 High Energy: Ultraviolet (UV) Low Energy: Infrared (IR)

Soret band 420 nm or g-band Mb Fe(II) ------Mb Fe(II) + CO ------

Visible region Visible bands Q bands a-band, b-band

b a

3 H2O/OH- O2 CO

Fe(III) Fe(II) Fe(II) Fe(II) Soret band at 420 nm His His His His metHb deoxy Hb Oxy Hb Carbon monoxy Hb metMb deoxy Mb Oxy Mb Carbon monoxy Mb

H2O/ O2-Substrate CO Substrate Soret band at 450 nm Fe(III) Fe(II) Fe(II) Fe(II) Cytochrome P450 Cys Cys Cys Cys Active form

4 Monooxygenase Reactions by P450 (CYP)

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

RH: Hydrophobic (lipophilic) compounds, organic compounds, insoluble in water

ROH: Less hydrophobic and slightly soluble in water.

Drug in liver

ROH + GST → R-GS GST: glutathione S-

ROH + UGT → R-UG UGT: glucuronosyltransferaseGlucuronic acid

Insoluble compounds are converted into highly hydrophilic (water soluble) compounds. 5

Drug metabolism at liver: Sleeping pill, pain killer (Narcotic), etc.

Synthesis of hormones (steroidgenesis) at , brain, , intestine, lung,

Animal (Mammalian, Fish, Bird, Insect), Plants, Fungi, Bacteria

6 NSAID: non-steroid anti-inflammatory drug

7 8 9 10 11

Cytochrome P450: Cysteine-S binding to Fe(II) heme is important for activation of O2. Cytochrome c, Cytochrome b5: Electron-transfer relating heme proteins.

Myoglobin and hemoglobin: Histidine-midazole binding to Fe(II) heme is important for O2 storage and O2 carrier, respectively. 14 NADPH-P450 Reductase

Difference spectra: substrate binding 450 nm

Shunt reaction

NADPH-P450 Reductase

15 R: substrate R: substrate

Shunt reaction

R: substrate

R: substrate

R: substrate

16 17 Family Function Members Names CYP1 drug and steroid (especially ) metabolism 3 subfamilies, 3 , 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 ) metabolism 1 subfamily, 4 genes, 2 pseudogenes CYP3A4, CYP3A5, CYP3A7, CYP3A43 CYP4 or 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 CYP5A1 CYP7 biosynthesis 7-alpha hydroxylase of steroid nucleus 2 subfamilies, 2 genes CYP7A1, CYP7B1 CYP8 varied2 subfamilies, 2 genes CYP8A1 (), 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 CYP19steroid biosynthesis: 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 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 of 24-hydroxycholesterol 1 subfamily, 1 gene CYP39A1 CYP46 24-hydroxylase 1 subfamily, 1 gene CYP46A1 CYP51 cholesterol biosynthesis 1 subfamily, 1 gene, 3 pseudogenes CYP51A1 ( 14-alpha )

18 Fish, Crab and Bird P450s

Those animals are used for monitoring environmental contamination/pollution with using liver.

19 One of PCBs

Environmental chemicals: P450’s substrates Expression of P-glycoprotein and PCB, chloroethane, benzo[A] pyrene cytochrome P450 1A in intertidal fish , Insecticides (Anoplarchus purpurescens) exposed to environmental contaminants 20 Arctic Monitoring and Assessment Programme

21 Insects

Alkaloids are toxic for insects.

Growth regulation: molting Sex, Alarm, Group hormones Those are natural products of plants and used for insecticides.

P450 is involved in pheromone synthesis, fighting against plant , and fighting against insecticides. 22 An insect-specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis Proc. Nat. Acad. Sci. U.S.A. 109, 14858 (2012)

Abstract Insects use hydrocarbons as cuticular waterproofing agents and as contact pheromones. Although their biosynthesis from fatty acyl precursors is well established, the last step of hydrocarbon biosynthesis from long-chain fatty aldehydes has remained mysterious. We show here that insects use a P450 of the CYP4G family to oxidatively produce hydrocarbons from aldehydes. Oenocyte-directed RNAi knock-down of Drosophila CYP4G1 or NADPH-cytochrome P450 reductase results in flies deficient in cuticular hydrocarbons, highly susceptible to desiccation, and with reduced viability upon adult emergence. The heterologously expressed enzyme converts C18-trideuterated octadecanal to C17- trideuterated heptadecane, showing that the insect enzyme is an oxidative decarbonylase that catalyzes the cleavage of long-chain aldehydes to hydrocarbons with the release of carbon dioxide. This process is unlike cyanobacteria that use a nonheme diiron decarbonylase to make alkanes from aldehydes with the release of formate. The unique and highly conserved insect CYP4G are a key evolutionary innovation that allowed their colonization of land. 23 Fig. 1. Hydrocarbon biosynthesis from very long-chain fatty acyl thioesters in cyanobacteria and in insects. The decarbonylase enzyme from plants has not been formally identified to date. ACP, acyl carrier .

24 Fig. 2. Colocalization of CYP4G1 and CPR in oenocytes. Whole-mount immunocytochemistry of NADPH-cytochrome P450 reductase (Upper Left, FITC) and CYP4G1 (Upper Right, Alexa 633) in Drosophila abdomens. Confocal microscopy shows the bands of large oenocytes where both enzymes are colocalized (Lower Right, yellow). Lower Left is the bright field image showing bristles for scale. 25 Fig. 4. Desiccation resistance of adult D. melanogaster. The time course of adult male (▲) and female (●) fly survival in dry conditions is shown for control insects (full lines) and for flies with RNAi-suppressed CYP4G1 expression (stippled lines). n = 20 for each condition.

26 27 Butterfly •Citrus (lemon, orange) Leaves. Strong flavor. Flavonoids. •Green caterpillar of the Lime Butterfly can eat citrus leaves. •P450s of butterfly metabolize the compounds.

28 Plants

Synthesis of natural products

29 Plants Fights against insects and herbicides

30 31 Flower color was changed by manipulation of P450 genes: Rose

Blue Rose: Suntory

32 Flower color was changed by manipulation of P450 genes: Petunia

33 Bacterial P450s

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 solved by X-ray crystallography.

Very stable, easy to analyze, thus used as a model for P450 catalysis.

Mycobacterium tuberculosis P450s: Inhibitors : structure and functions 34 Biomimicry in reverse. The natural P450 monooxygenase enzyme catalyzes C–H bond oxidation. Through judicious choice of enzyme and substrate followed by directed evolution, Coelho et al. have re-engineered this protein to perform a different catalytic reaction, namely cyclopropanation.

Science 339, 283 (2013)

35 Olefin Cyclopropanation via Carbene Transfer Catalyzed by Engineered Cytochrome P450 Enzymes

Transition metal–catalyzed transfers of carbenes, nitrenes, and oxenes are powerful methods for functionalizing C=C and C–H bonds. Nature has evolved a diverse toolbox for oxene transfers, as exemplified by the myriad monooxygenation reactions catalyzed by cytochrome P450 enzymes. The isoelectronic carbene transfer to olefins, a widely used C–C bond–forming reaction in organic synthesis, has no biological counterpart. Here we report engineered variants of cytochrome P450BM3 that catalyze highly diastereo- and enantioselective cyclopropanation of styrenes from diazoester reagents via putative carbene transfer. This work highlights the capacity to adapt existing enzymes for the catalysis of synthetically important reactions not previously observed in nature.

Science 339, 307 (2013)

36 Science 339, 307 (2013) Fig. 1 (Left) Canonical mode of reactivity of cytochrome P450s. Monooxygenation of olefins and C-H bonds to and alcohols catalyzed by the ferryl porphyrin radical intermediate (compound I). (Right) Artificial mode of formal carbene transfer activity of cytochrome P450s, using diazoester reagents as carbene precursors.

37 Oxygen deprivation followed by reoxygenation causes pathological responses in many disorders, including ischemic stroke, attacks, and reperfusion injury. Key aspects of ischemia-reperfusion can be modeled by a Caenorhabditis elegans behavior, the O2-ON response, which is suppressed by hypoxic preconditioning or inactivation of the O2-sensing HIF (hypoxia-inducible factor) hydroxylase EGL-9. From a genetic screen, we found that the cytochrome P450 CYP-13A12 acts in response to the EGL-9–HIF-1 pathway to facilitate the O2-ON response. CYP-13A12 promotes oxidation of polyunsaturated fatty acids into , signaling molecules that can strongly affect inflammatory pain and ischemia- reperfusion injury responses in mammals. We propose that roles of the EGL-9– HIF-1 pathway and cytochrome P450 in controlling responses to reoxygenation after anoxia are evolutionarily conserved.

Science 341, 554 (2013)

38 Science 341, 557 (2013)

39 Morphinan biosynthesis in opium poppy requires a P450- fusion protein

Morphinan alkaloids from the opium poppy are used for pain relief. The direction of metabolites to morphinan biosynthesis requires isomerization of (S)- to (R)-reticuline. Characterization of high-reticuline poppy mutants revealed a genetic , designated STORR [(S)- to (R)-reticuline] that encodes both cytochrome P450 and oxidoreductase modules, the latter belonging to the aldo-keto reductase family. Metabolite analysis of mutant alleles and heterologous expression demonstrate that the P450 module is responsible for the conversion of (S)-reticuline to 1,2- dehydroreticuline, whereas the oxidoreductase module converts 1,2- dehydroreticuline to (R)-reticuline rather than functioning as a P450 redox partner. Proteomic analysis confirmed that these two modules are contained on a single polypeptide in vivo. This modular assembly implies a selection pressure favoring substrate channeling. The fusion protein STORR may enable microbial-based morphinan production.

Science 349, 309 (2015)

40 41 P450 Oxidoreductase Defi ciency: A Disorder of Steroidogenesis with Multiple Clinical Manifestations

Sci. Signal. 5, pt11 (2012)

Cytochrome P450 enzymes catalyze the biosynthesis of steroid hormones and metabolize drugs. There are seven human type I P450 enzymes in mitochondria and 50 type II enzymes in . Type II enzymes, including both drug-metabolizing and some steroidogenic enzymes, require electron donation from a two-fl avin protein, P450 oxidoreductase (POR). Although knockout of the POR gene causes embryonic lethality in mice, we discovered human POR deficiency as a disorder of steroidogenesis associated with the Antley-Bixler skeletal malformation syndrome …. nearly wild-type activity with P450c21, CYP1A2, and CYP2C19. Activity of a particular POR variant with…. POR deficiency is a newly described disorder of steroidogenesis, and POR variants may account for some genetic variation in drug metabolism.

42 CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

Nat. Med. 22, 278 (2016)

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasimesenchymal (QM- PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to inhibitors and . Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine- like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell–autonomous detoxification mechanism that must be overcome to prevent drug resistance. 43