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

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Presentation on theme: "Enolase Bridging Project John A. Gerlt Enzyme Function Initiative (EFI) Advisory Committee Meeting November 30, 2011."— Presentation transcript:

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

2 Capping Domain: Specificity Barrel Domain: Acid/base chemistry Enolase superfamily: partition of specificity and 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 2.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 2.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 e-80  35% seq.id.e-184  70% seq.id. Boundaries between functions: ManD

9 ≥ 70% sequence identity: functional significance ?

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

11 Structures

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

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 2.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 PGIPFK FBP Aldolase TIMG3PDHPGKPGMEnolase Rank (%)0.90.10.30.10.80.50.030.2 PDB HM 64% to 2cxr 1pfk HM 40% to 3elf 7tim HM 58% to 1nqa HM 45% to 1vpe HM 49% to 1ejj 1ebj 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. Operon docking: retrospective glycolysis

18 2PMQ gene cluster (Pelagibaca bermudensis)

19 Transporter: A Trp “cage” for a betaine

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

21 12345 Docked with 4-hydroxy proline Top 5 docking hits 2PMQ: Docking, a small active site

22 4-OH Pro betaine k cat /K m = 4300 M -1 s -1 Experimental testing of 2PMQ prediction Pro betaine k cat /K m = 380 M -1 s -1 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 2.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 3DIP 2NQL 1RVK 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 1. Cosmid library (from S. Farrand, UIUC) 2. Identification of dehydratase cosmids 3. Wanner mutagenesis of cosmids in E. coli 4. Transformation and recombination of mutant cosmids into A. tumefaciens C58 5.Phenotypic analyses (BioLog) 6.Metabolomics to discover pathways Phenotypic/metabolomic analyses by Micro Core

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

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