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Partial thesis defence presentation Presentation Transcript

  • 1. Structural Biology of Cytochrome P450 monooxygenases in Arabidopsis thaliana phenylpropanoid pathway Sanjeewa Rupasinghe Mary Schuler lab Department of Cell and Developmental Biology spring 2007
  • 2. Cytochrome P450 monooxygenases Use molecular O 2 and NADPH to catalyze monooxygenation reactions R-H + O 2 + NADPH  R-OH + H 2 O + NADP Catalyze key steps in metabolic pathways from archaea to mammals Responsible for drug metabolism in humans Responsible for insecticide metabolism in insects wavelength (nm)
  • 3. P450 genomes
    • Archaebacteria 6
    • Bacteria
    • (~ 50 species includes Mycobacterium
    • and Streptomyces) 430
    • Neurospora crassa 41
    • Saccharomyces cerevisiae (baker’s yeast) 3
    • Drosophila melanogaster (fruit fly) 84
    • Caenorhabditis elegans (nematode) 74
    • Xenopus laevis at least 41
    • Danio rerio (zebrafish) at least 81
    • Mus musculus (mouse) 102
    • Homo sapiens (human) 57
    • Conifers 311
    • Arabidopsis thaliana 272
    • Oryza sativa (rice) 323
  • 4. Roles of plant P450s Specialized Biosynthetic P450s Defense Toxins Phytoalexins Furanocoumarins Cyanogenic Glucosides Alkaloids Glucosinolates Monoterpenes/Diterpenes
    • Specialized Detoxicative P450s
      • Herbicides
      • Sulfonylureas
      • Phenylureas
      • Insecticides
      • Pollutants
    General Biosynthetic P450s Phenylpropanoids Lignins Flavonoids Anthocyanins Hormones Gibberellins Jasmonic acid Auxin Brassinosteroids Fatty acid Cutins/suberins
  • 5. Arabidopsis P450s (Bak and Paquette, 2001)
  • 6. phenylpropanoid P450s OH OH O OH O CYP73A5 OH O OH OH OH OH O OH OH CYP84A1 O O OH OH OH OH O O OH OH OH OH OH CYP75B1 O O OH COOH OH OH O O OH COOH OH OH OH CYP98A3
  • 7. CYP98A3 p -coumaroyl-shikimate-3-hydroxylase
    • Biosynthesis of lignin monomers.
        • Mutant (ref8) has reduced lignin content
        • Composed of non wild type monomers
        • (Franke et al., 2002)
    • Synthesis antioxidants and bioactive esters
    • chlorogenic acid (coffee cup quality)
    • rosmarinic acid
    • caffeoyltyramine
    • (Mahesh et al., 2005)
    • Synthesis of flavour compounds
        • vanillin
        • gingerol
        • capsaicin
        • safrole
        • eugenol
  • 8. Structural biology of P450s in A. thaliana phenylpropanoid pathway Homology modeling and substrate docking Mutagenesis and protein engineering X-ray crystallography
  • 9.
    • 32 unique crystal structure templates are available
      • 12 from class II
      • 20 from class I
      • 2 from class III
    Improving P450 homology modeling
  • 10. CYP102 from Bacillus megaterium RMSD 5.8 Å
  • 11. CYP102 from Bacillus megaterium RMSD 5.3 Å
  • 12. Improving P450 homology modeling Class-independent alignment Single template Hybrid template 2.5 2.3 1.2 1.2 4.7 4.6 5.8 5.3 4.4 4.4 CYP2C5 CYP2C8 CYP3A4 CYP102 CYP158A2 Class-dependent alignment Single template Hybrid template 2.0 2.0 1.2 1.2 3.9 3.8 5.6 5.4 3.9 3.6
  • 13. Applications of P450 homology modeling
      • Substrate specificity
        • Phenylpropanoid P450s
        • (Rupasinghe et al 2003)
        • Fatty acid hydroxylases
        • (Rupasinghe et al 2007)
        • parsnip webworm, black swallowtail, corn earworm P540s
        • (Mao et al; 2006, Wen et al; 2006 - Collaboration with Berenbaum lab UIUC )
      • P450 reductase interactions
        • parsnip earworm CYP6AB3
        • (Mao et al; 2007- Collaboration with Berenbaum lab UIUC)
      • P450 structural stability
        • Streptomyces coelicolor CYP157C1
        • (Rupasinghe et al; 2006- Collaboration with Waterman lab, Vanderbilt University
      • High throughput docking to find the functionality of orphan P450s
  • 14. Kyoto encyclopedia of Genes and Genomes ( www.kegg.com ) 11000 + biological compounds Other databases 3000+ compounds List of most possible substrates Arabidopsis thaliana P450s 272 + genes Functions known for only 40 http://arabidopsis-p450.biotec.uiuc.edu Homology modeling High throughput docking
  • 15. Development of an E. coli- based functional reconstitution system for Arabidopsis P450s
    • Yeast co- expression system
      • Arabidopsis P450 reductase constitutively expressed.
    • Sf9 insect cell co-expression system
    • Problems of the systems
      • Low P450 yield
      • No P450 expression/express in 420 form.
      • Expensive and requires expertise
  • 16.
    • ATR1 has not been expressed in E coli
    • ATR2 expressed in a truncated form
    • P450 purification
      • Ni –ion affinity
      • Anion exchange
    • ATR2 purification
      • 2’5’-adenosine diphosphate affinity
    Development of an E coli based functional reconstitution system for Arabidopsis P450s 455 380
  • 17. Development of an E coli based functional reconstitution system for Arabidopsis P450s
    • Functional reconstitution
    • protein purification
    • p -coumaroyl shikimate hydroxylation assay
  • 18. Protein engineering in CYP98A3 CYP98A3 CYP73A5 O O OH OH O OH OH O OH OH O OH OH O O 292 302 381 382 473 479 301 309 368 375 484 489 1 CYP98A3 CYP73A5
  • 19. X-ray crystallographic studies of phenylpropanoid P450s
    • Gene cloning
      • CYP98A3 - gift form Dr. Danièle Werck-Reichhart (CNRS France)
      • CYP73A5- cloned from Arabidopsis total RNA
      • CYP84A1- cloned from Arabidopsis root RNA
      • CYP75B1- cloned from Arabidopsis silique mRNA
    • Requirements for P450 crystallography
      • soluble protein
      • large amounts
      • Clean homogeneous preparations
  • 20. X-ray crystallographic studies of phenylpropanoid P450s CYP98A3 CYP98A3wt MSWFLIAVATIAAVVSYKLI QRLRYKFPPG PSPKPIVGNLYDIKP VRFRCYYE CYP98A3D1 MA------------------QRLRYKFPPGPSPKPIVGNLYDIKPVRFRCYYE CYP98A3D2 MA------------------QR R R R KFPPGPSPKPIVGNLYDIKPVRFRCYYE promoter/ E. coli strain
  • 21. X-ray crystallographic studies of phenylpropanoid P450s CYP73A5 CYP73A5wt MDLLLLEKSLIAVFVAVILATVI SKLRGKKLKL PPGPIPIP IFGNWLQVGDDL CYP73A5D1 MA---------------------SKLRGKKLKLPPGPIPIPIFGNWLQVGDDL
  • 22. CYP98A3 and CYP73A5 crystallization screening Collaboration with David Stout Lab (Scripps Research Institute )
    • More than 2000 conditions were screened
    • Molecular Dimensions Ltd
      • MemStart & MemSys HT96
      • PACT TM
    • Emerald Biosystems
      • Wizard 1 &2
    • Hampton Research
      • Index HT
      • Crystal Screen
    CYP98A3 CYP98A3 CYP98A3 CYP73A5
  • 23. Structural Biology of P450s in A. thaliana phenylpropanoid pathway Homology modeling Mutagenesis and Protein engineering X-ray crystalography NMR
  • 24. Solid state NMR studies of Phenylpropanoid P450s Collaboration with Chad Rienstra lab (UIUC) Labeling P450s with C 13 and N 15 (Rupasinghe et al submitted to chem biochem journal) Promoter / Chaperone co-expression System P450 yield (mg/L of culture) Minimum Media Terrific Broth tac promoter ND 3 Bacteriopage T7 promoter ND 3 tac promoter / araB groES-groEL 4 8 tac promoter / Pzt1 groES-groEL 2 6 Spectra acquired by Heather Fererics , Chad Rienstra lab
  • 25. Acknowledgments
    • Schuler Lab (UIUC)
    • Dr. Mary A. Schuler
    • Dr. Shajahan Ali
    • Dr. Mitten Bilgin
    • Dr. Hui Duan
    • Dr. Wenfu Mao
    • Dr. Zhimou Wen
    • Dr. Ting-Lan Chiu
    • Iyoncy Rodrigo
    • Cindy McDonnell
    • Goudong Niu
    • Kathryn Palacio
    • Devi Annamalai
    • Amy Dunlap
    • Reed Johnson
    • Chine mee Lai
    • Junaid Begwala
    • Kara Sandnfort
    • 3D-Visualization Laboratory (UIUC)
    • Dr. Jerome Baudry (Vizlab)
    • Werck-Reichchart Lab (CNRS Stratsbrogh France)
    • Dr. Danile Werck-Reichchart
    • Dr. Pascaline Ullman
    • Berenbaum Lab (UIUC)
    • Dr. May Berenbaum
    • Dr. Art Zangal
    • Dr. Eva Castells
    • Sligar Lab (UIUC)
    • Dr. Steven Sligar
    • Brad Baas
    • Tom Makris
    • Rienstra lab (UIUC)
    • Dr. Chad Rienstra
    • Heather Frerics
    • Deb Berthold
    • Ying Li
    Berhow Lab (USDA IL) Dr. Mark Berhow Ray Holloway Johnson Lab (Scripps) Dr. Eric Jonson Stout Lab (Scripps) Dr. David Stout Committee members Dr. David Clayton Dr. Byron Kempher Dr. Phil Newmark Dr. May Berenbaum Suported by NIH R01-GM71826 grant NSF MCB0115068 grant