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Enolase Bridging Project John A. Gerlt Enzyme Function Initiative (EFI) Advisory Committee Meeting

Enolase Bridging Project John A. Gerlt Enzyme Function Initiative (EFI) Advisory Committee Meeting November 30, 2011. Enolase superfamily: partition of specificity and chemistry. Capping Domain: Specificity. Barrel Domain: Acid/base chemistry.

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Enolase Bridging Project John A. Gerlt Enzyme Function Initiative (EFI) Advisory Committee Meeting

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  1. Enolase Bridging Project John A. Gerlt Enzyme Function Initiative (EFI) Advisory Committee Meeting November 30, 2011

  2. Enolase superfamily: partition of specificity and chemistry Capping Domain: Specificity Barrel Domain: Acid/base chemistry

  3. Enolase superfamily: > 20 assigned functions

  4. Current directions 1. Superfamily/Genome, Protein/Structure, and Computation Cores: boundaries between functions ManD, GlucD, TarD, MR Computation Core: 2PMQ by operon docking 3. Microbiology Core: New pathways and functions, focusing on Agrobacterium tumefaciens

  5. Current directions 1. Superfamily/Genome, Protein/Structure, and Computation Cores: boundaries between functions ManD, GlucD, TarD, MR Computation Core: 2PMQ by operon docking 3. Microbiology Core: New pathways and functions, focusing on Agrobacterium tumefaciens

  6. SSN: dehydratases in EN superfamily Mandelate racemase D-Glucarate dehydratase D-Mannonate dehydratase Galactarate dehydratase D-Arabinonate dehydratase Galactonate dehydratase Gluconate dehydratase D-Tartrate dehydratase L-Fuconate dehydratase Galactarate/L-talarate dehydratase L-Rhamnonate dehydratase Unknown

  7. SSN: dehydratases in EN superfamily D-Mannonate dehydratase ManD

  8. Boundaries between functions: ManD e-80 ≥ 35% seq.id. e-184 ≥ 70% seq.id.

  9. ≥ 70% sequence identity: functional significance ?

  10. Activities: ManD, low ManD and/or GluD, none

  11. Structures

  12. Conserved active site structures 2QJJ 3BSM 3DFH 3GY1

  13. Conserved structures, except for active site loop: protein-protein interactions ? 2QJJ 3BSM 3DFH 3GY1

  14. Current directions 1. Superfamily/Genome, Protein/Structure, and Computation Cores: boundaries between functions ManD, GlucD, TarD, MR Computation Core: 2PMQ by operon docking 3. Microbiology Core: New pathways and functions, focusing on Agrobacterium tumefaciens

  15. Unknown family in the MLE subgroup: 2PMQ MLE MLEII MLE 2 OSBS NSAR NSAR 2 Dipeptide epimerase Unknown

  16. 2PMQ: Structure with No Function (SNF) from PSI-2

  17. Operon docking: retrospective glycolysis C. Kalyanaraman and M. P. Jacobson. "Studying enzyme-substrate specificity in silico: A case study of the E. coli glycolysis pathway”, Biochemistry, 49 (2010) 4003-4005.

  18. 2PMQ gene cluster (Pelagibaca bermudensis)

  19. Transporter: A Trp “cage” for a betaine

  20. Dioxygenase/hydroxylase: Homologues use aromatics Closest liganded homologues: 60% with 3N0Q, unliganded 18% with 1O7N: a naphthalene dioxygenase, cocrystalized with indole smaller active site: indole in 1O7N has lots of steric clashes Template: 1O7N

  21. 2PMQ: Docking, a small active site Docked with 4-hydroxy proline Top 5 docking hits

  22. Experimental testing of 2PMQ prediction kcat/Km = 4300 M-1s-1 4-OH Pro betaine kcat/Km = 380 M-1s-1 Pro betaine Genome context was helpful, but structures were essential First amino acid racemase in EN superfamily

  23. Proposed pathway for 4-OH Pro utilization Metabolomics to confirm pathway is in progress

  24. Current directions 1. Superfamily/Genome, Protein/Structure, and Computation Cores: boundaries between functions ManD, GlucD, TarD, MR Computation Core: 2PMQ by operon docking 3. Microbiology Core: New pathways and functions, focusing on Agrobacterium tumefaciens

  25. SSN: dehydratases in EN superfamily Mandelate racemase D-Glucarate dehydratase D-Mannonate dehydratase Galactarate dehydratase D-Arabinonate dehydratase Galactonate dehydratase Gluconate dehydratase D-Tartrate dehydratase L-Fuconate dehydratase Galactarate/L-talarate dehydratase L-Rhamnonate dehydratase Unknown

  26. Nine Agrobacterium tumefaciens dehydratases Four SNFs FucD 1RVK 3DIP 2NQL 3TJ4

  27. 1RVK: ordered 20s loop, large active site ?

  28. Library screening: 1RVK is a novel GlucD Agrobacterium utilize D-glucarate as carbon source ??

  29. Complex with L-lyxarohydroxamate

  30. Novel pathway for D-glucuronate catabolism ?

  31. Phenotypic/metabolomic analyses by Micro Core • Cosmid library (from S. Farrand, UIUC) • Identification of dehydratase cosmids • Wanner mutagenesis of cosmids in E. coli • Transformation and recombination of mutant cosmids into A. tumefaciens C58 • Phenotypic analyses (BioLog) 6. Metabolomics to discover pathways

  32. Current directions 1. Superfamily/Genome, Protein/Structure, and Computation Cores: boundaries between functions ManD, GlucD, TarD, MR Computation Core: 2PMQ by operon docking 3. Microbiology Core: New pathways and functions, focusing on Agrobacterium tumefaciens

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