Transcript P450
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: – – • “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 http://www.plantphysiol.org/cgi/content/abstract/110/3/1035 • Flavonoid-3’,5’-hydroxylase genes 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.