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Cytochrome P450 Monooxygenases

Cytochrome P450 Monooxygenases. William Green 2/17/11. Background. P450s are a group of heme -containing enzymes that utilize NADPH and/or NADH to produce functionalized organic substrates All P450s contain a highly conserved motif found near the C terminus: F- -G-R-C-G

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Cytochrome P450 Monooxygenases

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  1. Cytochrome P450 Monooxygenases William Green 2/17/11

  2. Background • P450s are a group of heme-containing enzymes that utilize NADPH and/or NADH to produce functionalized organic substrates • All P450s contain a highly conserved motif found near the C terminus: F- -G-R-C-G • Cytochrome P450-dependent monooxygenases“mediate a wide range of oxidative reactions involved in the biosynthesis of plant secondary metabolites including phenylpropanoids and phytoalexins” (Frank 1996). • Superfamily containing more than 450 CYP sequences among at least 65 gene families • P450 proteins are anchored to the ER membrane by “a hydrophobic helix near the N-terminus with most of the protein residing on the cytosolic face of the membrane" (Chapple 1998).

  3. Background Cont. • Two groups of P450s: • “Group A”: thought to have evolved since the divergence of plants from common ancestor • “Non-group A”: mostly closer to animal and fungal enzymes than group A proteins • Nomenclature: • Relationships between P450s determined by amino acid sequence identity • Genes >40% identical are said to be in the same P450 family (ex. CYP71 or CYP73) • If 40% or less, the new sequence defines a new family • Plant P450 genes begin with CYP71 through CYP99 • However recently these identifications have been exhaused and have seen been added to • If in the same family, but share < 55% identity they are said to be in either of two subfamilies (ex. CYP74A or CYP74B) • If > 55% they are in the same subfamily (ex. CYP71C includes CYP71C1 - 5) • Genes encoding proteins from different species are often members of the same subfamily • If > or = 97% identity, they represent allelic variants of the same gene

  4. Some Known P450s in Plants

  5. Focus of Study • First looked at highly conserved amino acids in heme binding domain • Two main pathways of P450 monooxygenasese: • Biosynthetic Pathway: • Leads to syntheiss of: UV protectants (flavonoids), anthocyanins, isoflavonoids/phytoalexins, fatty acids, hormones, signaling molecules, carotenoids, terpenes (defense compounds), and lignins (structural compounds) • Detoxification Pathway: • Drugs, carcinogens, herbicides and insecticides are all detoxified by P450 controlled hydroxylations • Cinnamate-4-Hydroxylase (C4H) was the first P450 to be characterized • C4H is encoded by the CYP73A subfamily • C4H catalyzes the para-hydroxylation of trans-cinnamic acid (derived from phenylalanine) • Known for involvment in phenylpropanoid pathway • Helps produce secondary metabolites: lignin, flavonoids, hydroxycinnamic acid esters, lignans, stilbenes, etc.

  6. Results • Blasted P450 genes against Blueberry databases • CYP73A1 mRNA from Helianthus tuberosus(Jerusalem artichoke) • CYP73A3 mRNA from Medicagotruncatula(Alfalfa) • CYP73A5 mRNA from Arabidposis thaliana • CYP73A9mRNA from PisumSativum(pea) • CYP82 mRNA from VitisVinifera(grape) • C4H mRNA from Selaginellamoellendorffii

  7. Results Cont. • 1 Hit found • CYP73A9: evalue (4e-115) • Blasted sequence against 454 to obtain BB sequence • 80% identity • Blasted against Illumina; 100% match with 454 • 0 hits in EST database

  8. Implications & Direction • Gene encoding C4H is in blueberry (possible ortholog of CYP73A9) • Want to examine heme binding domain • Flavonoid-3’,5’-hydroxylase genes http://www.plantphysiol.org/cgi/content/abstract/110/3/1035

  9. Sources • Chapple, Clint. "Molecular-Genetic Analysis Of Plant Cytochrome P450-Dependent Monooxygenases." Annual Review of Plant Physiology and Plant Molecular Biology 49.1 (1998): 311-43. Annual Review. Web. 11 Feb. 2011. http://www.annualreviews.org/doi/ pdf/10.1146/annurev.arplant.49.1.311. • Frank, Michael R., Joy M. Deyneka, and Mary A. Schuler. "Cloning of Wound-Induced Cytocrome P450 Monooxygenases Expressed in Pea." Plant Physiology 110.3 (1996): 1035-046. PLANT PHYSIOLOGY®. American Society of Plant Biologists. Web. 09 Feb. 2011. http://www.plantphysiol.org/cgi/content/abstract/110/3/1035.

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