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A Simple Anaylsis of Phenylpropanoid Pathway From the point of view of structure

A Simple Anaylsis of Phenylpropanoid Pathway From the point of view of structure. teammates: Liu Lanxuan Yin Jianmin. procedure. introduction of pathway and enzymes preparation of materials homology modelling protein-ligand docking protein-protein docking

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A Simple Anaylsis of Phenylpropanoid Pathway From the point of view of structure

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  1. A Simple Anaylsis of Phenylpropanoid Pathway From the point of view of structure teammates: Liu Lanxuan Yin Jianmin

  2. procedure • introduction of pathway and enzymes • preparation of materials • homology modelling • protein-ligand docking • protein-protein docking • summary and scope

  3. introduction • pathway: • phenylpropanoid pathway • enzymes: • cytochrome P450 and other enzyems

  4. Vogt, T. (2009). "Phenylpropanoid Biosynthesis." Molecular Plant 3(1): 2-20.

  5. The plant shikimate pathway is the entry to the biosynthesis ofphenylpropanoids. phenylpropanoid derived metabolites comprise and contribute to multiple biosynthetic branches other than lignin and flavonoid biosynthesis only Vogt, T. (2009). "Phenylpropanoid Biosynthesis." Molecular Plant 3(1): 2-20.

  6. The phenylpropanoid pathwayprovides pre-cursors for lignin in Arabidopsis source:KEGG

  7. In the last decade, knockout mutations and RNAi-mediatedsuppression in Arabidopsis and other model plants have facilitated the identification and functional characterization of genes and enzymes

  8. enzymes • cytochrome P450: • CYP84A1:ferulic acid 5-hydroxylase • CYP73A5:trans-cinnamate 4-monooxygenase • CYP98A3:coumaroylquinate(coumaroylshikimate) 3'-monooxygenase • other enzymes: • HCT:hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase • PAL:phenylalanine ammonia-lyase • 4CL:4-coumarate--CoA ligase • ...

  9. cytochrome P450 • Plant systems utilize a diverse array of cytochrome P450 monooxygenases (P450s) in their biosynthetic and detoxicative pathways. Those P450s in biosynthetic pathways play critical roles in the synthesis of lignins, UV protectants, pigments, defense compounds, fatty acids, hormones, and signaling molecules. Thosein catabolic pathways participate in the breakdown of endogenous compounds andtoxic compounds encountered in theenvironment. Schuler, M. A. and D. Werck-Reichhart (2003). "Functional genomics of P450s." Annual Review of Plant Biology 54: 629-667.

  10. At present,5100 sequences of plant cytochrome P450s(CYPs) have been annotated and named.The 5100 sequences include 3651 CYPs from 11 fully sequenced plant genomes:Arabidopsis, papaya, grapevine, soybean, tomato, rice,Brachypodium distachyon, Selaginella moellendorffii(lycopod), Physcomitrella patens (moss), Chlamydomonas reinhardtii and Volvox carteri (colonial green algae)An additional 1449 CYP sequences are named from 255 incompletely sequenced plant species with 1–183 sequences • Named sequences can be found at the CytochromeP450 Homepage: http://drnelson.uthsc.edu/CytochromeP450.html Nelson, D. and D. Werck-Reichhart (2011). "A P450-centric view of plant evolution." Plant Journal 66(1): 194-211.

  11. Meunier, B., S. P. de Visser, et al. (2004). "Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes." Chemical Reviews 104(9): 3947-3980

  12. Schoch, G., S. Goepfert, et al. (2001). "CYP98A3 from Arabidopsis thaliana is a 3 '-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway." Journal of Biological Chemistry 276(39): 36566-36574

  13. prepare materials • after searching PDB database, we found only one single enzyme(CAD) that has 3D structrue(based on Arabidopsis),in order to perform homology modelling we downloaded the sequences of CYP98A3,CYP73A5,CYP84A1 in the phenlypropanoid pathway of Arabidopsis

  14. sequence • >cyp73a5 • MDLLLLEKSLIAVFVAVILATVISKLRGKKLKLPPGPIPIPIFGNWLQVGDDLNHRNLVDYAKKFGDLFLLRMGQRNLVVVSSPDLTKEVLLTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQNREGWEFEAASVVEDVKKNPDSATKGIVLRKRLQLMMYNNMFRIMFDRRFESEDDPLFLRLKALNGERSRLAQSFEYNYGDFIPILRPFLRGYLKICQDVKDRRIALFKKYFVDERKQIASSKPTGSEGLKCAIDHILEAEQKGEINEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQSKLRNELDTVLGPGVQVTEPDLHKLPYLQAVVKETLRLRMAIPLLVPHMNLHDAKLAGYDIPAESKILVNAWWLANNPNSWKKPEEFRPERFFEEESHVEANGNDFRYVPFGVGRRSCPGIILALPILGITIGRMVQNFELLPPPGQSKVDTSEKGGQFSLHILNHSIIVMKPRNC • cyp98a3 • cyp84a1 • HCT • PAL • 4CL • ...

  15. sequence alignment of CYP98A3/CYP84A1/CYP73A5 Low sequence similarity high structure conservation

  16. homology modelling • blast search • sequence analysis • load templates • creating model • align model to template

  17. blast search cyp98a3 as example

  18. loaded templates structures 3RUKA 3QZ1A 3QM4A

  19. create models among several model,we pick the single one that has the high- est score as our result

  20. align structure to templates CCoAOMT 36% PAL 15.7%

  21. protein-ligand docking • method:CDOCKER(DS) • materials: • CYP73A5+substrate • CYP98A3+substrate • CYP84A1+substrate

  22. ligands Rupasinghe, S., J. Baudry, et al. (2003). "Common active site architecture and binding strategy of four phenylpropanoid P450s from Arabidopsis thaliana as revealed by molecular modeling." Protein Engineering 16(10): 721-731

  23. binding strategy Rupasinghe, S., J. Baudry, et al. (2003). "Common active site architecture and binding strategy of four phenylpropanoid P450s from Arabidopsis thaliana as revealed by molecular modeling." Protein Engineering 16(10): 721-731

  24. SRS sequence alignment Rupasinghe, S., J. Baudry, et al. (2003). "Common active site architecture and binding strategy of four phenylpropanoid P450s from Arabidopsis thaliana as revealed by molecular modeling." Protein Engineering 16(10): 721-731

  25. CDOCKER cyp98a3: site 10 cyp73a5: site2

  26. protein-protein docking • method:ZDOCK(DS) • materials: • CYP98A3 PAL • CYP73A5 HCT • CYP84A1 CCR CAD CCR...

  27. protein-protein interacion with cytochrome P450 • CYP450-CYP450 • CYP450-CPR • CYP450 N terminus • ER • CPR C terminus Subramanian, M., H. Tam, et al. (2010). "CYP2C9-CYP3A4 Protein-Protein Interactions: Role of the Hydrophobic N Terminus." Drug Metabolism and Disposition 38(6): 1003-1009. Subramanian, M., M. Low, et al. (2009). "CYP2D6-CYP2C9 Protein-Protein Interactions and Isoform-Selective Effects on Substrate Binding and Catalysis." Drug Metabolism and Disposition 37(8): 1682-1689. Localization of Cinnamic Acid 4-Monooxygenase and theMembrane-bound Enzyme System for Dhurrin Biosynthesisin Sorghum Seedlings' Received for publication May 11, 1977 and in revised form June 27, 1977JAMES A. SAUNDERS, ERIC E. CONN, CHIN Ho LIN, AND MIKIO SHIMADA3Department of Biochemistry and Biophysics, University of California, Davis, California 95616

  28. potential protein-protein interaction of cytochrome P450 in phenylpropanoid pathway • C4H-PAL • C3H-HCT • C4H-F5H • F5H-COMT Localization of Cinnamic Acid 4-Monooxygenase and theMembrane-bound Enzyme System for Dhurrin Biosynthesisin Sorghum Seedlings' Received for publication May 11, 1977 and in revised form June 27, 1977JAMES A. SAUNDERS, ERIC E. CONN, CHIN Ho LIN, AND MIKIO SHIMADA3Department of Biochemistry and Biophysics, University of California, Davis, California 95616 Bassard, J.-E., J. Mutterer, et al. (2012). "A novel method for monitoring the localization of cytochromes P450 and other endoplasmic reticulum membrane associated proteins: a tool for investigating the formation of metabolons." FEBS Journal 279(9): 1576-1583. Fraser (2011). "The Phenylpropanoid Pathway in Arabidopsis." The Arabidopsis Book

  29. cyp84a1-PAL cyp73a5-PAL

  30. pi-cation HBond pi-pi pi-sigma CYP7A35-PAL (pose99_Cluster9)

  31. CYP84A1-HCT CYP98A3-HCT CYP73A5-HCT • CYP84A1-PAL CYP98A3-PAL CYP73A5-PAL • CYP84A1-CCR CYP98A3-CCR CYP73A5-CCR • CYP84A1-CAD CYP98A3-CAD CYP73A5-CAD • CYP84A1-4CL CYP98A3-4CL CYP73A5-4CL others are still running...

  32. summary and scope summary: (1)catalysis mechanism of CYP450 (2)binding strategy of CYP450 and its substrate (3)potential protein-protein interaction of phenylpropanoid pathway (4)necessary docking(CDOCKER/ZDOCK)

  33. summary and scope • scope: (1)analyze the docking models to find something interesting and valuable (2)perform how protein-protein interaction affect the catalysis mechanism of CYP450 (3)write a paper

  34. 项目分工： 文献挖掘+PPT制作：刘岚轩 同源建模+对接模型：尹建民 Thank You!!

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