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Isoprenoid Synthase Bridging Project Dale Poulter Enzyme Function Initiative (EFI)

Isoprenoid Synthase Bridging Project Dale Poulter Enzyme Function Initiative (EFI) Advisory Committee Meeting November 30, 2011. IS Superfamily: Assigned Functions. Coupling. Cyclization. Chain Elongation. Cyclopropanation/Branching. Rearrangement. Structures for Coupling Enzymes.

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Isoprenoid Synthase Bridging Project Dale Poulter Enzyme Function Initiative (EFI)

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  1. Isoprenoid Synthase Bridging Project Dale Poulter Enzyme Function Initiative (EFI) Advisory Committee Meeting November 30, 2011

  2. IS Superfamily: Assigned Functions Coupling Cyclization Chain Elongation Cyclopropanation/Branching Rearrangement

  3. Structures for Coupling Enzymes Chain Elongation Cyclopropanation/Branching Rearrangement

  4. Structures for Cyclase Enzymes Pentalenene Synthase Streptomyces sp. 5-epi-Aristolochene Synthase Nicotiana tabacum Squalene-hopene Cyclase Alicyclobacillus acidocaldarius Taxadiene Synthase Taxus brevifolia

  5. IS Superfamily Squalene/Phytoene Synthase Like Polyprenyl Synthase Like

  6. Interactions with Core Facilities • Superfamily/Genome – identify structural/functional clusters in superfamily; • target selection – Patsy Babbitt; Daniel Almonacid • Protein – screen for activity, determine substrates/products – Steve Almo; • Brndan Hillerich; Ron Seidel • Structure – synthesize unreactive analogues; analyze structures – Steve Almo; • Yury Patskovsky; Matt Vetting • Computation – Docking/homology modeling for prediction of function – Matt • Jacobson; Frank Wallrapp • Microbiology – Identify operons containing isoprene synthase genes; identify • products of operon; in vivo function – John Cronan; McKay Wood

  7. Thiodiphosphate Analogues 100 mg each of C5-C20 analogues G. Ramamoorthy

  8. Aza Analogues G. Ramamoorthy

  9. IS Enzymes Requested – 307 - Chain elongation: 205 - Cyclopropanation/rearrangement: 66 - Cyclase: 16 (14 microbial; 2 plant) - Amino acid/aromatic: 20 Received – 98 (unique enzymes) - Chain elongation: 85 - Cyclopropanation/rearrangement: 7 - Cyclase: 6 (all microbial) Function assigned – 81 - Chain elongation: 78 - Cyclopropanation/rearrangement: 1 - Cyclase: 2 (microbial) X-ray structures – 21 (all chain elongation)

  10. TLC scans for samples 900393 and 900470 900470 900393 Std. C15 C20 C25 900393 - Z. mobilis squalene synthase 900470 – H. pylori FPP synthase C50 J.J. Pan G. Ramamoorthy IPP-DMAPP = 4:1 Std. Std. 900470 900393 Std. 900470 900393 Std. 900470 900393 Std. 900470 900393 900470 900393 C15 C15 C15 C20 C15 C20 C15 C20 C25 C20 C25 C20 C25 C25 C25 C50 C50 C50 C50 C50 IPP-DMAPP = 4:1 IPP-DMAPP = 4:1 IPP-DMAPP = 4:1 IPP-DMAPP = 4:1 IPP-DMAPP = 4:1

  11. Helicobacter pylori Farnesyl Diphosphate Synthase Yeast Squalene synthase -also synthesizes a C20 diphosphate from GPP J.J. Pan G. Ramamoorthy

  12. Zymomonas mobilis Squalene Synthase (hpnD) hpnA – hopanoid-associated sugar epimerase hpnB – hopanoid-associated glycosyltransferase hpnC – squalene synthase “like” protein hpnD – squalene synthase ‘like” protein hpnE – squalene-associated FAD-dependent desaturase hpnF – squalene-hopene cyclase hpnA hpnB hpnC hpnD hpnE hpnF ? Yeast Squalene synthase J.J. Pan G. Ramamoorthy

  13. IS Superfamily: Chain Elongation/Cyclopropanation D. Almonacid

  14. Chain Elongation/Cyclopropanation Interface Z. mobilis’ squalene synthase H. pylori’s FPP synthase A. tridentata’s chrysanthemyl diphosphate synthase D. Almonacid

  15. Chain Elongation/Cyclopropanation Interface D. Almonacid

  16. IS Superfamily: Chain Elongation/Cyclization D. Almonacid

  17. Chain Elongation/Cyclization Interface D. Almonacid

  18. Chain Elongation/Cyclization Interface D. Almonacid

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