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Cytochrome P450 Monooxygenases

Cytochrome P450 Monooxygenases. William Green Leland Taylor 3/29/11. Background. P450s are a group of heme-containing enzymes that utilize NADPH and/or NADH to produce functionalized organic substrates

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Cytochrome P450 Monooxygenases

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  1. Cytochrome P450 Monooxygenases William Green Leland Taylor 3/29/11

  2. Background • P450s are a group of heme-containing enzymes that utilize NADPH and/or NADH to produce functionalized organic substrates • 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). • All P450s contain a highly conserved Amino Acid motif found near the C terminus: F- -G-R-C-G • 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

  3. Goals of Study • Identify Homologs of p450s in the Blueberry genome • Create a network of pathways based on hits • Find key p450s involved in secondary metabolites • Ex. CYP73A5, CYP75A1, ect. • Isolate exons & introns • Ideal World: • Annotate whole genes • Link p450s with unknown functions to those with known functions • Comparisons of dicot vs. monocot (Arabidopsis, rice, blueberry, etc.) in order to improve phylogenetic understanding

  4. p450 hits against 454 database

  5. Isolate the sequence from scaffolds >scaffold02252|4452:4790|CYP51G2|2e-40 ACGTGGCCGGTCTTCGGCGGGTTAATCCGGTTCctAAAAGGaCCGGTTGTGATGCTAAGAGAAGAGTACCCGAAACTAGGGAGTGTGTTCACGTTGAACTTGTTCAACAAAAAGATCACTTTCTTTATCGGGCCTGAGGTTTCAGTCCATTTCTTTAAAGCTTCTGAATCTAATCTGAGCCAACAAGAAGTGTACAAGTACATTGTGCCTACCTTTGGGCCCGGAGTGATTTACGATGTTGATTATAGCGTACGGCAGGAGCAGTTTCGGTTCTTTACTGAGAGTCTAAGGGTTAACAAGTTGAAAGGTTATGTGGGTCAGATGCTTATTGAAGCTCA >scaffold02252|8063:8362|CYP51G2|8e-35 TCGCCTGCTTTCGCTAATCgTCTCCCTCATATATTCAAGGATCCGGACACGTTTGACCCTGACAGATTTGCTCCCGGTAGGGAagAAGACAAAGTGTCTGGGGCgTTttCGTACATCTCTTTTGGCGGTGGCAGGCACGCGTGTCTAGGGGAGCCGTTCGCGTATCTGCAAATAAAATCgATTTGGAGCCATTTGCTGAGGAACTTTGAGCTGGAACTGGTTTCACCTTTTCCGGAGATTGACTGGAATGCCATGGTCGTGGGTGTGAAAGGAAaGgTTATGGTGCGGTACAAGCGCCG >scaffold06866|14030:14566|CYP51G2|6e-16 ACTGAATCTTTTTGTACTTCCAAATTACAGGACATGTTtGTGGGAGGAaCTGATACAACCTCAGCAACCATGGAGTGGGCAATGGCAGAGCTGATGAAAAaTCCAACAAaGATgAaGAAAGCTCAAAAaGAGGTGCGAAGAGTGGTTGGGAAAAAaTCCAAGAtcAGTCAAGATGATATTGCTCAAATGGAATACTTAAAaTGTGTTGTCAAAGAgACTCTTAGATTACATCCTCCAGCTCCTTTTTtGGTtCCTAGAGAATTGACTGAGAGTACTACATTGGAAGGCTATGATATTCCTCCAAAAaCAAGAATATTCATTAATGCATGGGCAATTCAAAGagATTCCAAATACTGGGACCAGCCAGAAGAATTCcTTCCAGAGAGATTtGCTAACAACTTGGTTGATTTtAGAGGCCAAGAcTTCCAaTTCATTCCATTTGGAGCAGGGCgAAGGGGCTGTCCTGGCATTTCATTTGGTGTCATGGAAGTTGAACTTGTACTTGCCAACCTCTTGTACTGGTTTGACTGGAAGTT >scaffold06866|9256:9750|CYP51G2|6e-15 GCAGGAACTGATACAACTTCAACAACCTtAGAGTGGGCAATGGCAGAGCTAATGAAAAGTCCAACTAGGATGAAGAAAaTtCAAGAaGAGGTACGAAGaGTGGTtGGgAAGAAaTCCAAGATCAATCAAgaTGATATtGATCAAATGgACTACTTAAAATGTGTTGTCAAAGAGACTCTTAGATTACATCCTCCAGGTCCTTTTTtGGTtCCTAGAGAATCAAGTGAGAGTACTAAATTGGGAGGTTATGATATTCCTCCAAAAACAAGAATTTTCATCAATGTTTGGGCAATTCAAAGAGATCCCAAATACTGGGATAGGCCCGAAGAATTTCTTCCAGAGAGATTTGCTAACAACCCGATTGATTTtAAAGGCAAAGACTTCGAATTCATCCCATTTGGTGCAGGGCGAAGGGGCTGTCCTGGAATTTCATTTGCAATCACTGAAACTGAACAAGTGCTTGCCAATCTCTTGTACTGGTTTGATTGGAAGTT

  6. Align isolated sequences with EST >scaffold02252|4452:4790|CYP51G2|2e-40 ACGTGGCCGGTCTTCGGCGGGTTAATCCGGTTCctAAAAGGaCCGGTTGTGATGCTAAGAGAAGAGTACCCGAAACTAGGGAGTGTGTTCACGTTGAACTTGTTCAACAAAAAGATCACTTTCTTTATCGGGCCTGAGGTTTCAGTCCATTTCTTTAAAGCTTCTGAATCTAATCTGAGCCAACAAGAAGTGTACAAGTACATTGTGCCTACCTTTGGGCCCGGAGTGATTTACGATGTTGATTATAGCGTACGGCAGGAGCAGTTTCGGTTCTTTACTGAGAGTCTAAGGGTTAACAAGTTGAAAGGTTATGTGGGTCAGATGCTTATTGAAGCTCA Query= scaffold02252|8063:8362|CYP51G2|8e-35 > Contig9662 Query 4 CCTGCTTTCGCTAATCGTCTCCCTCATATATTCAAGGATCCGGACACGTTTGACCCTGAC 63 ||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||| Sbjct 582 CCTGCTTTCGCTAATCGTCTCCCTCATATTTTCAAGGATCCGGACACGTTTGACCCTGAC 641 Query 64 AGATTTGCTCCCGGTAGGGAAGAAGACAAAGT-GTCTGGGGCGTTTTCGTACATCTCTTT 122 ||||||||||||||||| |||||||||||||| | ||||||| ||||||||||||||||| Sbjct 642 AGATTTGCTCCCGGTAGAGAAGAAGACAAAGTAG-CTGGGGCATTTTCGTACATCTCTTT 700 Query 123 TGGCGGTGGCAGGCACGCGTGTCTAGGGGAGCCGTTCGCGTATCTGCAAATAAAATCGAT 182 ||| ||||||||||||| |||| |||||||||| || || ||||||||||||||| | || Sbjct 701 TGGTGGTGGCAGGCACGGGTGTTTAGGGGAGCCTTTTGCTTATCTGCAAATAAAAGCCAT 760 Query 183 TTGGAGCCATTTGCTGAGGAACTTTGAGCTGGAACTGGTTTCACCTTTTCCGGAGATTGA 242 ||||||||||||||||||||| ||||||||||||||||| |||||||||||||||||||| Sbjct 761 TTGGAGCCATTTGCTGAGGAATTTTGAGCTGGAACTGGTCTCACCTTTTCCGGAGATTGA 820 Query 243 CTGGAATGCCATGGTCGTGGGTGTGAAAGGAAAGGTTATGGTGCGGTACAAGCG 296 |||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 821 CTGGAATGCCATGGTCGTGGGTGTGAAAGGAAAGGTTATGGTGCGGTACAAGCG 874

  7. Issues/Frustrations • >scaffold07086|5306:4950|CYP51G2|6e-45 • Could not find good software… • JCVI, Washington University Genome Center • Blast has “tool” to isolate… poor documentation • Artemis stinks

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