Biodiversity of P-450 monooxygenase:

Cross-talk between chemistry and biology

Heme Fe(II)-CO complex 450 nm, different from those of hemoglobin and other heme proteins 410-420 nm. Cytochrome Pigment of 450 nm Cytochrome P450 CYP??? 1 Monooxygenase Reactions by Cytochromes 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 metabolism in liver

ROH + GST → R-GS GST: glutathione S-transferase

ROH + UGT → R-UG UGT: glucuronosyltransferaseGlucuronic acid

Insoluble compounds are converted into highly hydrophilic (water soluble) compounds. 2

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

Synthesis of steroid hormones (steroidgenesis) at adrenal cortex, brain, kidney, intestine, lung,

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

3 4 5 NSAID: non-steroid anti-inflammatory drug

6 7 8 Myoglobin: O2 storage Muscle Sashimi さしみ: Raw Fish Meat Fe(II) (Ferrous) heme Fe(III) (Ferric) heme, hemin Raw meat: Red: Tuna Protoporphyrin IX Raw meat: White: Flatfish

Hemoglobin: O2 carrier Blood 9

You need O2 because of continuous swimming. You don’t need O2. More myoglobin. Less myoglobin.

10

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. 11 NADPH-P450 Reductase

Difference spectra: substrate binding 450 nm

Shunt reaction

NADPH-P450 Reductase

12 R: substrate R: substrate

Shunt reaction

R: substrate

R: substrate

R: substrate

13 14 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 varied2 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 CYP19steroid 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)

15 Fish, Crab and Bird P450s

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

16 One of PCBs

Environmental chemicals: P450’s substrates Expression of P-glycoprotein and PCB, chloroethane, benzo[A] pyrene cytochrome P450 1A in intertidal fish Herbicides, Insecticides (Anoplarchus purpurescens) exposed to environmental contaminants 17 Arctic Monitoring and Assessment Programme

18 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 toxin, and fighting against insecticides. 19 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 enzyme 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 enzymes are a key evolutionary innovation that allowed their colonization of land. 20 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 protein.

21 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. 22 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.

23 24 Butterfly •Citrus (lemon, orange) Leaves. Strong flavor. Flavonoids. •Green caterpillar of the Lime Butterfly can eat citrus leaves. •P450s of butterfly metabolize the compounds.

25 Plants

Synthesis of natural products

26 Plants Fights against insects and herbicides

27 28 Flower color was changed by manipulation of P450 genes: Rose

Blue Rose: Suntory

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

30 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 protein structure 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 31