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 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).

Background Cont. Two groups of P450s: Nomenclature: “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

Some Known P450s in Plants

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.

Results Blasted P450 genes against Blueberry databases CYP73A1 mRNA from Helianthus tuberosus (Jerusalem artichoke) CYP73A3 mRNA from Medicago truncatula (Alfalfa) CYP73A5 mRNA from Arabidposis thaliana CYP73A9 mRNA from Pisum Sativum (pea) CYP82 mRNA from Vitis Vinifera (grape) C4H mRNA from Selaginella moellendorffii

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

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

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.