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