Cytochrome p450 monooxygenases
Download
1 / 17

Cytochrome P450 Monooxygenases - PowerPoint PPT Presentation


  • 305 Views
  • Uploaded on

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:

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Cytochrome P450 Monooxygenases' - damara


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
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." 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>.


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 *.

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*


Order orientation
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