Cytochrome P 450 Monooxygenases William Green Leland Taylor
- Slides: 17
Cytochrome P 450 Monooxygenases William Green Leland Taylor 4/12/11
Background • P 450 s are enzymes that produce functionalized organic substrates • 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 • Superfamily containing more than 450 CYP sequences among at least 65 gene families – All P 450 s contain a highly conserved Amino Acid motif found near the C terminus: F- -G-R-C-G
P 450 identification • Arabidopsis p 450 data set – http: //www. p 450. kvl. dk/ • t. Blastn 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
CYP 71 A 19 CYP 71 B 2 CYP 71 B 36 CYP 71 A 24 CYP 81 F 2 CYP 81 H 1 CYP 706 A 2 CYP 71 B 27 CYP 71 A 12 CYP 81 F 4 CYP 71 B 29 CYP 72 A 7 CYP 72 A 10 CYP 71 A 20 CYP 81 D 3 CYP 706 A 3 CYP 714 A 2 CYP 72 A 14 CYP 705 A 5 CYP 82 C 2 CYP 71 B 9 CYP 705 A 24 CYP 71 B 18 P CYP 714 A 1 CYP 90 A 1 CYP 71 A 28 CYP 71 B 21 CYP 71 B 15 CYP 715 A 1 CYP 705 A 15 CYP 90 B 1 CYP 97 C 1 CYP 77 B 1 CYP 90 C 1 CYP 705 A 20 CYP 89 A 7 CYP 79 F 2 CYP 89 A 4 CYP 79 B 2 CYP 86 C 4 CYP 79 C 1 CYP 86 A 2 CYP 96 A 4 CYP 86 C 1 CYP 79 F 1 CYP 702 A 2 CYP 94 C 1 CYP 716 A 1 CYP 79 A 4 P CYP 94 B 2 CYP 710 A 4 CYP 72 A 12 P CYP 87 A 3 P CYP 79 B 4 P Number of Hits in Illumina Database 160 140 120 100 80 60 40 20 0
CYP 71 A 22 CYP 71 A 18 CYP 81 D 2 CYP 81 F 3 CYP 81 H 1 CYP 71 B 19 CYP 81 D 4 CYP 75 B 1 CYP 706 A 1 CYP 71 B 11 CYP 81 D 7 CYP 82 G 1 CYP 71 B 7 CYP 71 A 16 CYP 76 C 3 CYP 71 B 29 CYP 82 C 3 CYP 77 A 7 CYP 705 A 34 P CYP 71 B 22 CYP 71 B 8 CYP 81 D 11 CYP 705 A 27 CYP 72 A 9 CYP 72 A 15 CYP 71 B 32 CYP 707 A 4 CYP 714 A 1 CYP 709 B 2 CYP 90 C 1 CYP 89 A 5 CYP 735 A 1 CYP 705 A 22 CYP 72 A 13 CYP 76 C 8 P CYP 88 A 3 CYP 73 A 5 CYP 88 A 4 CYP 704 A 1 CYP 97 B 3 CYP 86 C 1 CYP 96 A 3 CYP 86 A 8 CYP 708 A 2 CYP 94 D 1 CYP 79 A 4 P CYP 94 B 1 CYP 705 A 14 P CYP 94 B 2 CYP 94 D 3 P CYP 74 A CYP 79 B 4 P Number of Hits in 454 Database 350 300 250 200 150 100 50 0
320 300 280 260 240 220 200 454 180 160 140 120 100 80 60 40 20 0 0 20 40 60 80 100 120 140 160 Illumina 180 200 220 240 260 280 300 320
P 450 s of Interest • CYP 74 B 2 – Hydroperoxide lyase – Electron carrier/heme binding – Fatty acid metabolism • Possibly related to Allene oxide synthase • Involved in the response to wounding • CYP 75 B 1 – Flavonoid 3’-monooxygenase – Involved in Flavonoid biosynthesis – Hydroxylated derivatives are common flower pigments in related species • CYP 73 A 5 – Cinnamate-4 -Hydroxylase – Involved in phenylpropanoid pathway • Lignin, flavonoids, lignans, stilbenes, etc.
Good Order • CYP 74 B 2 - Fatty Acid Hydroperoxide lyase 3217 3558 5928 6599 6992 7363 454 scaffold 12002 35 Arab. CYP 74 B 2 Query 145 368 490
P 450 s of Interest • CYP 74 B 2 – Hydroperoxide lyase – Electron carrier/heme binding – Fatty acid metabolism • Possibly related to Allene oxide synthase • Involved in the response to wounding • CYP 75 B 1 – Flavonoid 3’-monooxygenase – Involved in Flavonoid biosynthesis – Hydroxylated derivatives are linked to pigment color in other species • CYP 73 A 5 – Cinnamate-4 -Hydroxylase – Involved in phenylpropanoid pathway • Lignin, flavonoids, lignans, stilbenes, etc.
Order/Orientation Mixup • P 450 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 scaffold 00711 Arab. CYP 75 B 1 4 234 18277 18972 Rev Comp 502 438 / 426 294 273 28588 29010 Rev Comp 53
P 450 s of Interest • CYP 74 B 2 – Hydroperoxide lyase – Electron carrier/heme binding – Fatty acid metabolism • Possibly related to Allene oxide synthase • Involved in the response to wounding • CYP 75 B 1 – Flavonoid 3’-monooxygenase – Involved in Flavonoid biosynthesis – Hydroxylated derivatives are common flower pigments in related species • CYP 73 A 5 – Cinnamate-4 -Hydroxylase – Involved in phenylpropanoid pathway • Lignin, flavonoids, lignans, stilbenes, etc.
Problem Order • CYP 73 A 5 – Cinnamate-4 -Hydroxylase 44 454 scaffold 10240 195 53 298 448 294 296 383 345 Arab. CYP 73 A 5 812 1261 1869 2131 2442 3386 3679 5751 Rev. Com. 486 444 6458 6572 7018
Further Work • Create alignment with sequences of P 450 genes as root • Determine introns/exons & paralogs/alleles using alignment as guide • Comparison of determined P 450 s against other related species • Phylogenetic Distribution of P 450 s
Acknowledgements • Dr. Allan Brown • NC State University • David H. Murdock Research Institute • Dr. Malcolm Campbell & Davidson College
References Nelson, David R. "Mining Databases for Cytochrome P 450 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 Werck Reichhart and Soren Bak. "Comparative Genomics of Rice and Arabidopsis. Analysis of 727 Cytochrome P 450 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>.
Good Order Query: CYP 74 B 2 Z 97339, 4201 -7105 There is a 5 bp deletion in the genomic sequence from BAC Z 97339 as compared to the c. DNA AF 087932. This deletion produces a frame shift that gives the P 450 from the genomic sequence an inaccurate N-terminus. The deletion point is marked with a *. CYP 74 B 2 scaffold 12002 5 e-61 144 9 e-47 368 1 e-40 35 368 490 145 5928 6599 6992 7363 3217 3558 Arabidopsis Query Sequence: Fatty Acid Hydroperoxide lyase MLLRTMAATSPRPPPSTSLTSQQPPSPPSQLPLRTMPGSYGWPLVGPLSDRLDFQGPDKFFRTRAEKYKSTVFRTNIPPTFPFFG NVNPNIVAVLDVKSFSHLFDMDLVDKRDVLIGDFRPSLGFYGGVRVGVYLDTTEPKHAQIKGFAMETLKRSSKVWLQELRSNLN IFWGTIESEISKNGAASYIFPLQRCIFSFLCASLAGVDASVSPDIAENGWKTINTWLALQVIPTAKLGVVPQPLEEILLHTWPYPSLL IAGNYKKLYNFIDENAGDCLRLGQEEFGLTRDEAIQNLLFVLGFNAYGGFSVFLPSLIGRITGDNSGLQERIRTEVRRVCGSGSDLN FKTVNEMELVKSVVYETMRFSPPVPLQFARARKDFQISSHDAVFEVKKGELLCGYQPLVMRDANVFDEPEEFKPDRYVGETGSE LLNYLYWSNGPQTGTPSASNKQCAAKDIVTLTASLLVADLFLRYDTITGDSGSIKAVVKAK*
Order/Orientation >CYP 75 B 1_AF 155171_NTC gi|8132327|gb|AF 155171. 1|AF 155171 Arabidopsis thaliana flavonoid CYP 75 B 1 scaffold 00711 4 e-34 294 438 29010 28588 CYP 75 B 1 scaffold 00711 2 e-17 53 273 18972 18277 CYP 75 B 1 scaffold 00711 8 e-13 426 502 4 234 >scaffold 00711|4: 234|CYP 75 B 1|8 e-13 ATTGAa. TACAAAGGGCAACACTTTGa. GTTGATCCCATTt. GGATCTGGGag. AAGGATGTGTGTTGGCTt. CCCa. TTa. GCCCAGCGAGTGATTc. ACCTTG CTCTGGCc. ACCCTGATACAa. TCTTTTGATTGGGAGGTTAGCAGCTCTCTAGGAATt. ACAGACATGACTGAAAAGATGGGGATATCAGTGC GGAAGCTAGTACCa. CTAAAAGCAa. TACCCACAAAGCGA >scaffold 00711|REV_COMP|29010: 28588|CYP 75 B 1|4 e-34 AAAGGGAATCAACTCGAAATGTTGACCCTTGTATTCAATATTTGAGTCTAGAAACCTCTCGGg. CTTGAAAGACAAGGGGTCg. TCCCAGGCATCCG GGTc. TCt. CCCAATTGCCCATGCATTCACAAa. CACTTGTGTGCCTTt. GGGTATGAGATATCCCATGAACTTGGTATCCTCCATTGTGTTCCGGGGAAG g. AGAAg. AGGAAGTGCAGGGTGTAATCGg. AGGGTTt. CCTTCACCACGGCTt. GAAGATATGGCAATCGATCCATGTCGCTCTCCTCCACTTTCCTGTT CCGCCCAACTACTCGGTCGATTTCCTCCTGCAACTTTTTCATGCAGCGAGGg. TTt. CGTAATAACTCAGTGAAa. CCCCATTCGATGCTGATACTAGTT GTTTCTGACCCCGCGAAAAACATTTCCTGCAGCAATGA >scaffold 00711|REV_COMP|18972: 18277|CYP 75 B 1|2 e-17 CAACATAGCATCCAAGAAGTCTTTGTTCACCTTCTCAGTACCTGAATTCTTCTTCTCCTGAACCCTCTCCGTTACAAACTCTGCTACAATCTTCAGGG CTCGTCCCAAGTTCAGATCCATATCTCTTTTGAGCCCCATGGGGTCCATTTCAGAAATGGTAGGAAATCAGCTACGTTAGGCTTCCCTGATA CCACCATTATCATTTCCATTGCCTCGAGGAATTCACGCCCTTTGTCTGAGTTCGGTTCCAACAGATCTCTAGATAGCATAAGATTGCCTACCACATT GAAAGACATGAGGAAATGGCTTATCTGTACCTCTCCCGATCCTCCGCTTGCACGCGACGCCTGTGAATCCTCCTCGATCCACCGGATCA TGTTCTGGACACACTTCTCCCTGAGAGCAGCTGTACTGTTGACTCGTTTGGTGACCAGGAGCTCCGAGGAGCATATTCTCCTTAg. CAGTCGCCAAT ATGTGCCATATTTGCTCAGAGCAAGTGATCCTTGGTTGTAGCTGCACGATGTCATTACATCGGTGACATTTCGGTCGCAGAAACTTGAATCGTGG TTCTTGAAAAACTCTGCGGCTGCCTTGGGTGATTGTATAACCATGGTGTTTACCGATCCAAACTTAAACCaaa. TTACTGGTCCATAttt. GAATCTGAG CTTGTACATGTCTATGTGTGG
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