Cytochrome p450 monooxygenases
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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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Cytochrome p450 monooxygenases

Cytochrome P450 Monooxygenases

William Green

Leland Taylor

4/12/11


Background

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


P450 identification

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


P450s of interest

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.


Good order

Good Order

  • CYP74B2 - Fatty Acid Hydroperoxidelyase

3217 3558 5928 6599 6992 7363

454 scaffold12002

35 145 368 490

Arab. CYP74B2 Query


P450s of interest1

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.


Order orientation mixup

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


P450s of interest2

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.


Problem order

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.


Further work

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


Acknowledgements

Acknowledgements

  • Dr. Allan Brown

  • NC State University

  • David H. Murdock Research Institute

  • Dr. Malcolm Campbell & Davidson College


References

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 WerckReichhartand SorenBak. "Comparative Genomics of Rice andArabidopsis. Analysis of 727 Cytochrome P450 Genes and Pseudogenesfrom 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 order1

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 *.

CYP74B2scaffold120025e-6114436859286599

CYP74B2scaffold120029e-4736849069927363

CYP74B2scaffold120021e-403514532173558

Arabidopsis Query Sequence: Fatty Acid Hydroperoxidelyase

MLLRTMAATSPRPPPSTSLTSQQPPSPPSQLPLRTMPGSYGWPLVGPLSDRLDFQGPDKFFRTRAEKYKSTVFRTNIPPTFPFFGNVNPNIVAVLDVKSFSHLFDMDLVDKRDVLIGDFRPSLGFYGGVRVGVYLDTTEPKHAQIKGFAMETLKRSSKVWLQELRSNLNIFWGTIESEISKNGAASYIFPLQRCIFSFLCASLAGVDASVSPDIAENGWKTINTWLALQVIPTAKLGVVPQPLEEILLHTWPYPSLLIAGNYKKLYNFIDENAGDCLRLGQEEFGLTRDEAIQNLLFVLGFNAYGGFSVFLPSLIGRITGDNSGLQERIRTEVRRVCGSGSDLNFKTVNEMELVKSVVYETMRFSPPVPLQFARARKDFQISSHDAVFEVKKGELLCGYQPLVMRDANVFDEPEEFKPDRYVGETGSELLNYLYWSNGPQTGTPSASNKQCAAKDIVTLTASLLVADLFLRYDTITGDSGSIKAVVKAK*


Order orientation

Order/Orientation

>CYP75B1_AF155171_NTC gi|8132327|gb|AF155171.1|AF155171 Arabidopsis thaliana flavonoid

CYP75B1scaffold007114e-342944382901028588

CYP75B1scaffold007112e-17532731897218277

CYP75B1scaffold007118e-134265024234

>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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