Cytochrome P 450 Monooxygenases William Green Leland Taylor

Cytochrome P 450 Monooxygenases William Green Leland Taylor 3/29/11

Background • P 450 s are a group of heme-containing enzymes that utilize NADPH and/or NADH to produce functionalized organic substrates • Cytochrome P 450 -dependent monooxygenases “mediate a wide range of oxidative reactions involved in the biosynthesis of plant secondary metabolites including phenylpropanoids and phytoalexins” (Frank 1996). • All P 450 s contain a highly conserved Amino Acid motif found near the C terminus: F- -G-R-C-G • Two main pathways of P 450 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 P 450 controlled hydroxylations

Goals of Study • Identify Homologs of p 450 s in the Blueberry genome • Create a network of pathways based on hits • Find key p 450 s involved in secondary metabolites • Ex. CYP 73 A 5, CYP 75 A 1, ect. • Isolate exons & introns • Ideal World: • Annotate whole genes • Link p 450 s with unknown functions to those with known functions • Comparisons of dicot vs. monocot (Arabidopsis, rice, blueberry, etc. ) in order to improve phylogenetic understanding

p 450 hits against 454 database Query Hit Subject Hit E Value Query Start Query End Subject Start Subject End CYP 89 A 7_NTC scaffold 00007 7. 00 E-08 1303 1432 132874 132745 CYP 89 A 7_NTC scaffold 00007 3. 00 E-06 862 977 133386 133271 CYP 89 A 7_NTC scaffold 00007 4. 00 E-05 1207 1280 133008 132936 CYP 89 A 9_NTC scaffold 00007 3. 00 E-19 1300 1478 132877 132699 … . . . … … … . . . …

Isolate the sequence from scaffolds >scaffold 02252|4452: 4790|CYP 51 G 2|2 e-40 ACGTGGCCGGTCTTCGGCGGGTTAATCCGGTTCct. AAAAGGa. CCGGTTGTGATGCTAAGAGTACCCGAAACTAGGGAGTGTGTTCACGTT GAACTTGTTCAACAAAAAGATCACTTTATCGGGCCTGAGGTTTCAGTCCATTTCTTTAAAGCTTCTGAATCTGAGCCAACAAGAAG TGTACAAGTACATTGTGCCTACCTTTGGGCCCGGAGTGATTTACGATGTTGATTATAGCGTACGGCAGGAGCAGTTTCGGTTCTTTACTGAGAGT CTAAGGGTTAACAAGTTGAAAGGTTATGTGGGTCAGATGCTTATTGAAGCTCA >scaffold 02252|8063: 8362|CYP 51 G 2|8 e-35 TCGCCTGCTTTCGCTAATCg. TCTCCCTCATATATTCAAGGATCCGGACACGTTTGACCCTGACAGATTTGCTCCCGGTAGGGAag. AAGACAAAGT GTCTGGGGCg. TTtt. CGTACATCTCTTTTGGCGGTGGCACGCGTGTCTAGGGGAGCCGTTCGCGTATCTGCAAATAAAATCg. ATTTGGAGCC ATTTGCTGAGGAACTTTGAGCTGGAACTGGTTTCACCTTTTCCGGAGATTGACTGGAATGCCATGGTCGTGGGTGTGAAAGGAAa. Gg. TTATGGTG CGGTACAAGCGCCG >scaffold 06866|14030: 14566|CYP 51 G 2|6 e-16 ACTGAATCTTTTTGTACTTCCAAATTACAGGACATGTTt. GTGGGAGGAa. CTGATACAACCTCAGCAACCATGGAGTGGGCAATGGCAGAGCTGAT GAAAAa. TCCAACAAa. GATg. Aa. GAAAGCTCAAAAa. GAGGTGCGAAGAGTGGTTGGGAAAAAa. TCCAAGAtc. AGTCAAGATGATATTGCTCAAATGG AATACTTAAAa. TGTGTTGTCAAAGAg. ACTCTTAGATTACATCCTCCAGCTCCTTTTTt. GGTt. CCTAGAGAATTGACTGAGAGTACTACATTGGAA GGCTATGATATTCCTCCAAAAa. CAAGAATATTCATTAATGCATGGGCAATTCAAAGag. ATTCCAAATACTGGGACCAGAAGAATTCc. TTCC AGAGAGATTt. GCTAACAACTTGGTTGATTTt. AGAGGCCAAGAc. TTCCAa. TTCATTCCATTTGGAGCAGGGCg. AAGGGGCTGTCCTGGCATTTCAT TTGGTGTCATGGAAGTTGAACTTGTACTTGCCAACCTCTTGTACTGGTTTGACTGGAAGTT >scaffold 06866|9256: 9750|CYP 51 G 2|6 e-15 GCAGGAACTGATACAACTTCAACAACCTt. AGAGTGGGCAATGGCAGAGCTAATGAAAAGTCCAACTAGGATGAAGAAAa. Tt. CAAGAa. GAGGTACG AAGa. GTGGTt. GGg. AAGAAa. TCCAAGATCAAga. TGATATt. GATCAAATGg. ACTACTTAAAATGTGTTGTCAAAGAGACTCTTAGATTACATC CTCCAGGTCCTTTTTt. GGTt. CCTAGAGAATCAAGTGAGAGTACTAAATTGGGAGGTTATGATATTCCTCCAAAAACAAGAATTTTCATCAATGTT TGGGCAATTCAAAGAGATCCCAAATACTGGGATAGGCCCGAAGAATTTCTTCCAGAGAGATTTGCTAACAACCCGATTTt. AAAGGCAAAGA CTTCGAATTCATCCCATTTGGTGCAGGGCGAAGGGGCTGTCCTGGAATTTCATTTGCAATCACTGAACAAGTGCTTGCCAATCTCTTGT ACTGGTTTGATTGGAAGTT

Align isolated sequences with EST >scaffold 02252|4452: 4790|CYP 51 G 2|2 e-40 ACGTGGCCGGTCTTCGGCGGGTTAATCCGGTTCct. AAAAGGa. CCGGTTGTGATGCTAAGAGTACCCGAAACTAGGGAGTGTGTTCACGTT GAACTTGTTCAACAAAAAGATCACTTTATCGGGCCTGAGGTTTCAGTCCATTTCTTTAAAGCTTCTGAATCTGAGCCAACAAGAAG TGTACAAGTACATTGTGCCTACCTTTGGGCCCGGAGTGATTTACGATGTTGATTATAGCGTACGGCAGGAGCAGTTTCGGTTCTTTACTGAGAGT CTAAGGGTTAACAAGTTGAAAGGTTATGTGGGTCAGATGCTTATTGAAGCTCA Query= 63 Sbjct scaffold 02252|8063: 8362|CYP 51 G 2|8 e-35 > Contig 9662 4 CCTGCTTTCGCTAATCGTCTCCCTCATATATTCAAGGATCCGGACACGTTTGACCCTGAC |||||||||||||||||||||||||||||| 582 CCTGCTTTCGCTAATCGTCTCCCTCATATTTTCAAGGATCCGGACACGTTTGACCCTGAC Query 64 122 Sbjct 642 AGATTTGCTCCCGGTAGGGAAGAAGACAAAGT-GTCTGGGGCGTTTTCGTACATCTCTTT ||||||||| | |||||||||||| AGATTTGCTCCCGGTAGAGAAGAAGACAAAGTAG-CTGGGGCATTTTCGTACATCTCTTT Query 123 182 Sbjct 701 TGGCGGTGGCACGCGTGTCTAGGGGAGCCGTTCGCGTATCTGCAAATAAAATCGAT |||||||||| |||||||| | || TGGTGGTGGCACGGGTGTTTAGGGGAGCCTTTTGCTTATCTGCAAATAAAAGCCAT Query 183 242 Sbjct 761 TTGGAGCCATTTGCTGAGGAACTTTGAGCTGGAACTGGTTTCACCTTTTCCGGAGATTGA |||||||||||||||||||| TTGGAGCCATTTGCTGAGGAATTTTGAGCTGGAACTGGTCTCACCTTTTCCGGAGATTGA Query 243 Sbjct 821 Query CTGGAATGCCATGGTCGTGGGTGTGAAAGGTTATGGTGCGGTACAAGCG ||||||||||||||||||||||||||| CTGGAATGCCATGGTCGTGGGTGTGAAAGGTTATGGTGCGGTACAAGCG 296 874 641 700 760 820

Issues/Frustrations • >scaffold 07086|5306: 4950|CYP 51 G 2|6 e-45 • Could not find good software… – JCVI, Washington University Genome Center – Blast has “tool” to isolate… poor documentation • Artemis stinks