1. Cytochrome P450 Monooxygenases William Green Leland Taylor 4/12/11

2. Background P450s are heme-containing enzymes that produce functionalized organic substrates 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 Superfamily containing more than 450 CYP sequences among at least 65 gene families – All P450s contain a highly conserved Amino Acid motif found near the C terminus: F- -G-R-C-G

3. P450 identification Arabidopsis p450 data set – http://www.p450.kvl.dk/ http://www.p450.kvl.dk/ tBlastn against blueberry data (454 database) – 272 query’s – Multiple hits per query (identified by gene) Parse data to find partial/whole genes

7. P450s of Interest CYP74B2 – Hydroperoxide lyase – Electron carrier/heme binding – Fatty acid metabolism Possibly related to Allene oxide synthase Involved in the response to wounding CYP75B1 – Flavonoid 3’-monooxygenase – Involved in Flavonoid biosynthesis – Hydroxylated derivatives are common flower pigments in related species CYP73A5 – Cinnamate-4-Hydroxylase – Involved in phenylpropanoid pathway Lignin, flavonoids, lignans, stilbenes, etc.

8. Good Order CYP74B2 - Fatty Acid Hydroperoxide lyase 3217 3558 5928 6599 6992 7363 35 145 368 490 Arab. CYP74B2 Query 454 scaffold12002

9. P450s of Interest CYP74B2 – Hydroperoxide lyase – Electron carrier/heme binding – Fatty acid metabolism Possibly related to Allene oxide synthase Involved in the response to wounding CYP75B1 – Flavonoid 3’-monooxygenase – Involved in Flavonoid biosynthesis – Hydroxylated derivatives are common flower pigments in related species CYP73A5 – Cinnamate-4-Hydroxylase – Involved in phenylpropanoid pathway Lignin, flavonoids, lignans, stilbenes, etc.

10. Order/Orientation Mixup P450 linked to flavonoid – Visual results of hits on one scaffold N’s in between sequence. Must make a guess at orientation. This evidence shows it should be flipped 454 scaffold00711 Arab. CYP75B1 53 273 294 426 / 438 502 42342901028588 Rev Comp 1897218277 Rev Comp SEQ-442NSEQ-N-SEQ

11. P450s of Interest CYP74B2 – Hydroperoxide lyase – Electron carrier/heme binding – Fatty acid metabolism Possibly related to Allene oxide synthase Involved in the response to wounding CYP75B1 – Flavonoid 3’-monooxygenase – Involved in Flavonoid biosynthesis – Hydroxylated derivatives are common flower pigments in related species CYP73A5 – Cinnamate-4-Hydroxylase – Involved in phenylpropanoid pathway Lignin, flavonoids, lignans, stilbenes, etc.

12. Problem Order 454 scaffold10240 CYP73A5 – Cinnamate-4-Hydroxylase Arab. CYP73A5 812 1261 1869 2131 2442 3386 3679 5751 6458 6572 7018 44 195 298 448 53 294 296 383 486 345 444 Rev. Com.

13. Further Work Focus on other P450s of interest – CYP88A2: involved in gibberlin synthesis (growth factors) – Etc. Connect P450s with other gene pathways Develop phylogenetic distribution of P450s

14. Acknowledgements Dr. Allan Brown NC State University David H. Murdock Research Institute Dr. Malcom Campbell & Davidson College

15. References Nelson, David R. "Mining Databases for Cytochrome P450 Genes." Methodsin Enzymology 357 (2002): 3-15. Science Direct. Elsevier B.V., 16 Dec. 2003. Web. link Nelson, David R., Mary A. Schuler, Suzanne M. Paquette, Daniele Werck Reichhart and Soren Bak. "Comparative Genomics of Rice and Arabidopsis. Analysis of 727 Cytochrome P450 Genes and Pseudogenes from a Monocot and a Dicot." Plant Physiology 135.2 (2004): 756-72. PLANT PHYSIOLOGY®. American Society of Plant Biologists, 31 Mar. 2004. Web..