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Cytochrome P450 Monooxygenases

Cytochrome P450 Monooxygenases. William Green Leland Taylor 4/12/11. Background. P450s are enzymes that produce functionalized organic substrates Two main pathways of P450 monooxygenasese : Biosynthetic Pathway:

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Cytochrome P450 Monooxygenases

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

  2. Background • P450s are 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/ • tBlastn against blueberry data (454 database) • 272 query’s (identified by gene) • Multiple hits per query (separated by scaffold) • Average Gene Length ~500 Amino Acids • Parse data to find partial/whole genes

  4. P450s of Interest • CYP74B2 – Hydroperoxidelyase • 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.

  5. Good Order • CYP74B2 - Fatty Acid Hydroperoxidelyase 3217 3558 5928 6599 6992 7363 454 scaffold12002 35 145 368 490 Arab. CYP74B2 Query

  6. P450s of Interest • CYP74B2 – Hydroperoxidelyase • 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 linked to pigment color in other species • CYP73A5 – Cinnamate-4-Hydroxylase • Involved in phenylpropanoid pathway • Lignin, flavonoids, lignans, stilbenes, etc.

  7. 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 4 234 454 scaffold00711 18277 18972 28588 29010 Rev Comp Rev Comp 294 Arab. CYP75B1 438 / 426 273 53 502

  8. P450s of Interest • CYP74B2 – Hydroperoxidelyase • 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.

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

  10. Further Work • Create alignment with sequences of P450 genes as root • Determine introns/exons & paralogs/alleles using alignment as guide • Comparison of determined P450s against other related species • Phylogenetic Distribution of P450s

  11. Acknowledgements • Dr. Allan Brown • NC State University • David H. Murdock Research Institute • Dr. Malcolm Campbell & Davidson College

  12. References Nelson, David R. "Mining Databases for Cytochrome P450 Genes." Methods in 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 WerckReichhartand SorenBak. "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. <http://www.plantphysiol.org/content/135/2/756>.

  13. Good Order Query: CYP74B2 Z97339, 4201-7105 There is a 5bp deletion in the genomic sequence from BAC Z97339 as compared to the cDNA AF087932. This deletion produces a frame shift that gives the P450 from the genomic sequence an inaccurate N-terminus. The deletion point is marked with a *. CYP74B2 scaffold12002 5e-61 144 368 5928 6599 CYP74B2 scaffold12002 9e-47 368 490 6992 7363 CYP74B2 scaffold12002 1e-40 35 145 3217 3558 Arabidopsis Query Sequence: Fatty Acid Hydroperoxidelyase MLLRTMAATSPRPPPSTSLTSQQPPSPPSQLPLRTMPGSYGWPLVGPLSDRLDFQGPDKFFRTRAEKYKSTVFRTNIPPTFPFFGNVNPNIVAVLDVKSFSHLFDMDLVDKRDVLIGDFRPSLGFYGGVRVGVYLDTTEPKHAQIKGFAMETLKRSSKVWLQELRSNLNIFWGTIESEISKNGAASYIFPLQRCIFSFLCASLAGVDASVSPDIAENGWKTINTWLALQVIPTAKLGVVPQPLEEILLHTWPYPSLLIAGNYKKLYNFIDENAGDCLRLGQEEFGLTRDEAIQNLLFVLGFNAYGGFSVFLPSLIGRITGDNSGLQERIRTEVRRVCGSGSDLNFKTVNEMELVKSVVYETMRFSPPVPLQFARARKDFQISSHDAVFEVKKGELLCGYQPLVMRDANVFDEPEEFKPDRYVGETGSELLNYLYWSNGPQTGTPSASNKQCAAKDIVTLTASLLVADLFLRYDTITGDSGSIKAVVKAK*

  14. Order/Orientation >CYP75B1_AF155171_NTC gi|8132327|gb|AF155171.1|AF155171 Arabidopsis thaliana flavonoid CYP75B1 scaffold00711 4e-34 294 438 29010 28588 CYP75B1 scaffold00711 2e-17 53 273 18972 18277 CYP75B1 scaffold00711 8e-13 426 502 4 234 >scaffold00711|4:234|CYP75B1|8e-13 ATTGAaTACAAAGGGCAACACTTTGaGTTGATCCCATTtGGATCTGGGagAAGGATGTGTGTTGGCTtCCCaTTaGCCCAGCGAGTGATTcACCTTGCTCTGGCcACCCTGATACAaTCTTTTGATTGGGAGGTTAGCAGCTCTCTCTCTCTAGGAATtACAGACATGACTGAAAAGATGGGGATATCAGTGCGGAAGCTAGTACCaCTAAAAGCAaTACCCACAAAGCGA >scaffold00711|REV_COMP|29010:28588|CYP75B1|4e-34 AAAGGGAATCAACTCGAAATGTTGACCCTTGTATTCAATATTTGAGTCTAGAAACCTCTCGGgCTTGAAAGACAAGGGGTCgTCCCAGGCATCCGGGTcTCtCCCAATTGCCCATGCATTCACAAaCACTTGTGTGCCTTtGGGTATGAGATATCCCATGAACTTGGTATCCTCCATTGTGTTCCGGGGAAGgAGAAgAGGAAGTGCAGGGTGTAATCGgAGGGTTtCCTTCACCACGGCTtGAAGATATGGCAATCGATCCATGTCGCTCTCCTCCACTTTCCTGTTCCGCCCAACTACTCGGTCGATTTCCTCCTGCAACTTTTTCATGCAGCGAGGgTTtCGTAATAACTCAGTGAAaCCCCATTCGATGCTGATACTAGTTGTTTCTGACCCCGCGAAAAACATTTCCTGCAGCAATGA >scaffold00711|REV_COMP|18972:18277|CYP75B1|2e-17 CAACATAGCATCCAAGAAGTCTTTGTTCACCTTCTCAGTACCTGAATTCTTCTTCTCCTGAACCCTCTCCGTTACAAACTCTGCTACAATCTTCAGGGCTCGTCCCAAGTTCAGATCCATATCTCTTTTGAGCCCCATGGGGTCCATCCATTTCAGAAATGGTAGGAAATCAGCTACGTTAGGCTTCCCTGATACCACCATTATCATTTCCATTGCCTCGAGGAATTCACGCCCTTTGTCTGAGTTCGGTTCCAACAGATCTCTAGATAGCATAAGATTGCCTACCACATTGAAAGACATGAGGAAGAGGAAATGGCTTATCTGTACCTCTCCCGATCCTCCGCTTGCACGCGACGCCTGTGAATCCTCCTCGATCCACCGGATCATGTTCTGGACACACTTCTCCCTGAGAGCAGCTGTACTGTTGACTCGTTTGGTGACCAGGAGCTCCGAGGAGCATATTCTCCTTAgCAGTCGCCAATATGTGCCATATTTGCTCAGAGCAAGTGATCCTTGGTTGTAGCTGCACGATGTCATTACATCGGTGACATTTCGGTCGCAGAAACTTGAATCGTGGTTCTTGAAAAACTCTGCGGCTGCCTTGGGTGATTGTATAACCATGGTGTTTACCGATCCAAACTTAAACCaaaTTACTGGTCCATAtttGAATCTGAGCTTGTACATGTCTATGTGTGG

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