partial thesis defence presentation
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Structural Biology of Structural Biology of Cytochrome P450 monooxygenases in Cytochrome P450 monooxygenases in
Arabidopsis thalianaArabidopsis thaliana phenylpropanoid pathwayphenylpropanoid pathway
Sanjeewa RupasingheSanjeewa RupasingheMary Schuler labMary Schuler lab
Department of Cell and Developmental BiologyDepartment of Cell and Developmental Biology
spring 2007spring 2007
wavelength (nm)
Use molecular O2 and NADPH to catalyze monooxygenation reactions
R-H + O2 + NADPH R-OH + H2O + NADP
Catalyze key steps in metabolic pathways from archaea to mammals
Responsible for drug metabolism in humans
Responsible for insecticide metabolism in insects
Cytochrome P450 monooxygenases
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
Roles of plant P450s
Specialized Biosynthetic P450s
Defense Toxins Phytoalexins Furanocoumarins Cyanogenic Glucosides Alkaloids GlucosinolatesMonoterpenes/Diterpenes
Specialized Detoxicative P450s
Herbicides Sulfonylureas PhenylureasInsecticidesPollutants
General Biosynthetic P450s
Phenylpropanoids Lignins Flavonoids AnthocyaninsHormones Gibberellins Jasmonic acid Auxin Brassinosteroids Fatty acid Cutins/suberins
(Bak and Paquette, 2001)
Arabidopsis P450s
OH
OHOOHO
CYP73A5
OHO
OHOHOH
OHO
OH
OH
CYP84A1
O O
OH
OH
OH
OH
O O
OH
OH
OH
OH
OH
CYP75B1
O O
OH
COOHOH
OH
O O
OH
COOHOH
OH
OH
CYP98A3
phenylpropanoid P450s
CYP98A3p-coumaroyl-shikimate-3-hydroxylase
Biosynthesis of lignin monomers.
Mutant (ref8) has reduced lignin contentComposed of non wild type monomers
(Franke et al., 2002)
Synthesis antioxidants and bioactive esters
chlorogenic acid (coffee cup quality)rosmarinic acidcaffeoyltyramine
(Mahesh et al., 2005)
Synthesis of flavour compounds
vanillingingerolcapsaicinsafroleeugenol
Homology modeling andsubstrate docking
Mutagenesis and protein engineering
X-ray crystallography
Structural biology of P450s in A. thaliana phenylpropanoid pathway
32 unique crystal structure templates are available
12 from class II
20 from class I
2 from class III
Improving P450 homology modeling
RMSD 5.8 Å
CYP102 from Bacillus megaterium
RMSD 5.3 Å
CYP102 from Bacillus megaterium
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
Improving P450 homology modeling
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
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 40http://arabidopsis-p450.biotec.uiuc.edu
Homology modelingHigh throughput docking
Development of an E. coli-based functional reconstitution system for Arabidopsis P450s
Yeast co- expression system Yeast co- expression system Arabidopsis P450 reductase constitutively Arabidopsis P450 reductase constitutively
expressed.expressed.
Sf9 insect cell co-expression systemSf9 insect cell co-expression system
Problems of the systemsProblems of the systems Low P450 yieldLow P450 yield No P450 expression/express in 420 form.No P450 expression/express in 420 form. Expensive and requires expertiseExpensive and requires expertise
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 Development of an E coli E coli based functional based functional reconstitution system for reconstitution system for ArabidopsisArabidopsis P450s P450s
455
380
Development of an Development of an E coli E coli based functional based functional reconstitution system for reconstitution system for ArabidopsisArabidopsis P450s P450s
Functional reconstitution
protein purification
p-coumaroyl shikimate hydroxylation assay
O
O
OH
OHO
OH
OH
OOH
OHO
OH
OH
O O
CYP98A3 CYP73A5
292 302 381 382 473 479
301 309 368 375 484 489
1CYP98A3
CYP73A5
Protein engineering in CYP98A3Protein engineering in CYP98A3
X-ray crystallographic studies of X-ray crystallographic studies of phenylpropanoid P450s 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
CYP98A3CYP98A3wt MSWFLIAVATIAAVVSYKLIQRLRYKFPPGPSPKPIVGNLYDIKPVRFRCYYECYP98A3D1 MA------------------QRLRYKFPPGPSPKPIVGNLYDIKPVRFRCYYECYP98A3D2 MA------------------QRRRRKFPPGPSPKPIVGNLYDIKPVRFRCYYE
X-ray crystallographic studies of X-ray crystallographic studies of phenylpropanoid P450s phenylpropanoid P450s
promoter/E. coli strain
CYP73A5CYP73A5wt MDLLLLEKSLIAVFVAVILATVISKLRGKKLKLPPGPIPIPIFGNWLQVGDDLCYP73A5D1 MA---------------------SKLRGKKLKLPPGPIPIPIFGNWLQVGDDL
X-ray crystallographic studies of X-ray crystallographic studies of phenylpropanoid P450s phenylpropanoid P450s
CYP98A3 and CYP73A5 crystallization screeningCollaboration with David Stout Lab (Scripps Research Institute )
More than 2000 conditions were screened
Molecular Dimensions Ltd MemStart & MemSys HT96 PACTTM
Emerald Biosystems Wizard 1 &2
Hampton Research Index HT Crystal Screen
CYP98A3 CYP98A3
CYP98A3 CYP73A5
Homology modelingMutagenesis and
Protein engineeringX-ray crystalography
Structural Biology of P450s in A. thaliana phenylpropanoid pathway
NMR
Promoter / Chaperone co-Promoter / Chaperone co-expression Systemexpression System
P450 yield (mg/L of culture)P450 yield (mg/L of culture)
Minimum Minimum MediaMedia
Terrific BrothTerrific Broth
tac tac promoterpromoter NDND 33
Bacteriopage T7 promoterBacteriopage T7 promoter NDND 33
tactac promoter / promoter / araBaraB groES- groES-groELgroEL
44 88
tactac promoter / promoter / Pzt1Pzt1 groES- groES-groELgroEL
22 66
Labeling P450s with CLabeling P450s with C13 13 and Nand N1515
Spectra acquired by Heather Fererics , Chad Rienstra lab
Solid state NMR studies of Phenylpropanoid Solid state NMR studies of Phenylpropanoid P450sP450s
Collaboration with Chad Rienstra lab (UIUC)Collaboration with Chad Rienstra lab (UIUC)
(Rupasinghe et al submitted to chem biochem journal)
AcknowledgmentsAcknowledgmentsSchuler Lab (UIUC)Schuler Lab (UIUC)Dr. Mary A. SchulerDr. Mary A. Schuler
Dr. Shajahan AliDr. Shajahan AliDr. Mitten BilginDr. Mitten BilginDr. Hui DuanDr. Hui DuanDr. Wenfu MaoDr. Wenfu MaoDr. Zhimou WenDr. Zhimou WenDr. Ting-Lan ChiuDr. Ting-Lan Chiu
Iyoncy Rodrigo Iyoncy Rodrigo Cindy McDonnellCindy McDonnellGoudong NiuGoudong NiuKathryn PalacioKathryn PalacioDevi AnnamalaiDevi AnnamalaiAmy DunlapAmy DunlapReed JohnsonReed Johnson
Chine mee LaiChine mee LaiJunaid BegwalaJunaid BegwalaKara SandnfortKara Sandnfort
3D-Visualization Laboratory (UIUC)3D-Visualization Laboratory (UIUC)
Dr. Jerome Baudry (Vizlab)Dr. Jerome Baudry (Vizlab)
Werck-Reichchart Lab (CNRS Stratsbrogh France)Werck-Reichchart Lab (CNRS Stratsbrogh France)
Dr. Danile Werck-ReichchartDr. Danile Werck-Reichchart
Dr. Pascaline UllmanDr. Pascaline Ullman
Berenbaum Lab (UIUC)Berenbaum Lab (UIUC)
Dr. May BerenbaumDr. May Berenbaum
Dr. Art ZangalDr. Art Zangal
Dr. Eva CastellsDr. Eva Castells
Sligar Lab (UIUC)Sligar Lab (UIUC)
Dr. Steven SligarDr. Steven Sligar
Brad Baas Brad Baas
Tom Makris Tom Makris
Rienstra lab (UIUC) Rienstra lab (UIUC)
Dr. Chad RienstraDr. Chad Rienstra
Heather FrericsHeather Frerics
Deb BertholdDeb Berthold
Ying LiYing Li
Berhow Lab (USDA IL)Berhow Lab (USDA IL)Dr. Mark BerhowDr. Mark BerhowRay HollowayRay Holloway
Johnson Lab (Scripps)Johnson Lab (Scripps)Dr. Eric JonsonDr. Eric Jonson
Stout Lab (Scripps)Stout Lab (Scripps)Dr. David StoutDr. David Stout
Committee membersCommittee membersDr. David ClaytonDr. David ClaytonDr. Byron KempherDr. Byron KempherDr. Phil NewmarkDr. Phil NewmarkDr. May BerenbaumDr. May Berenbaum
Suported by Suported by NIH R01-GM71826 grant NIH R01-GM71826 grant NSF MCB0115068 grantNSF MCB0115068 grant