Partial thesis defence presentation

Structural Biology of Cytochrome P450 monooxygenases in Arabidopsis thaliana phenylpropanoid pathway Sanjeewa Rupasinghe Mary Schuler lab Department of Cell and Developmental Biology spring 2007

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)

P450 genomes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Roles of plant P450s Specialized Biosynthetic P450s Defense Toxins Phytoalexins Furanocoumarins Cyanogenic Glucosides Alkaloids Glucosinolates Monoterpenes/Diterpenes ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],General Biosynthetic P450s Phenylpropanoids Lignins Flavonoids Anthocyanins Hormones Gibberellins Jasmonic acid Auxin Brassinosteroids Fatty acid Cutins/suberins

Arabidopsis P450s (Bak and Paquette, 2001)

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

CYP98A3 p -coumaroyl-shikimate-3-hydroxylase ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Structural biology of P450s in A. thaliana phenylpropanoid pathway Homology modeling and substrate docking Mutagenesis and protein engineering X-ray crystallography

[object Object],[object Object],[object Object],[object Object],Improving P450 homology modeling

CYP102 from Bacillus megaterium RMSD 5.8 Å

CYP102 from Bacillus megaterium RMSD 5.3 Å

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

Applications of P450 homology modeling ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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

Development of an E. coli- based functional reconstitution system for Arabidopsis P450s ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Development of an E coli based functional reconstitution system for Arabidopsis P450s 455 380

Development of an E coli based functional reconstitution system for Arabidopsis P450s ,[object Object],[object Object],[object Object]

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

X-ray crystallographic studies of phenylpropanoid P450s ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

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

X-ray crystallographic studies of phenylpropanoid P450s CYP73A5 CYP73A5wt MDLLLLEKSLIAVFVAVILATVI SKLRGKKLKL PPGPIPIP IFGNWLQVGDDL CYP73A5D1 MA---------------------SKLRGKKLKLPPGPIPIPIFGNWLQVGDDL

CYP98A3 and CYP73A5 crystallization screening Collaboration with David Stout Lab (Scripps Research Institute ) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],CYP98A3 CYP98A3 CYP98A3 CYP73A5

Structural Biology of P450s in A. thaliana phenylpropanoid pathway Homology modeling Mutagenesis and Protein engineering X-ray crystalography NMR

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

Acknowledgments ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],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

Partial thesis defence presentation

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Partial thesis defence presentation