Cytochrome P450 Monooxygenases

William Green2/17/11

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

• Flavonoid-3’,5’-hydroxylase geneshttp://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.