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

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Enolase Bridging Project

John A. Gerlt

Enzyme Function Initiative (EFI)Advisory Committee Meeting

November 30, 2011

Capping Domain:Specificity

Barrel Domain:Acid/basechemistry

Enolase superfamily:partition of specificity and chemistry

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Enolase superfamily: > 20 assigned functions

-O2COPO3

CH2OHH

-O2COPO3

D-Glucarate

cis,cis-Muconate Muconolactone

OSBSHCHC

NH HAla

L-Ala-L-Glu L-Ala-D-Glu

AE Epim

Enolase

2-PGA PEP

OHOHOHOH

OHOHOH

D-Mannonate

HOHHOH

L-Idarate

antiCO2

O CO2-

N-Succ-L-Arg N-Succ-D-Arg

-O2C-O2C

3-Methyl Asp

CH3-O2C

Mesaconate

anti-NH3

HOHOHH

Galactarate

+H2NNH2 +H2N

(R)-Mandelate (S)-Mandelate

OHHHOH

D-Galactonate

OHOHHH

L-Rhamnonate

OHHOHOH

OHOHOH

D-Gluconate

HOHOHH

L-Fuconate

D-Tartrate

HOHOHH

Galactarate

GalrD/TalrD

HOHOHOH

L-Talarate

GalrD/TalrD

- H2O - H2O

D-Arabinonate

D-Xylonate

OHHOHOH

D-Glucarate

GlucD-II

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

Current directions

SSN: dehydratases in EN superfamilyMandelate racemase

D-Glucarate dehydrataseD-Mannonate dehydratase

Galactarate dehydrataseD-Arabinonate dehydratase

Galactonate dehydrataseGluconate dehydrataseD-Tartrate dehydratase

L-Fuconate dehydrataseGalactarate/L-talarate dehydratase

L-Rhamnonate dehydratase

Unknown

SSN: dehydratases in EN superfamily

D-Mannonate dehydratase

e-80 ≥ 35% seq.id. e-184 ≥ 70% seq.id.

Boundaries between functions: ManD

≥ 70% sequence identity: functional significance ?

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

Structures

2QJJ3BSM3DFH3GY1

Conserved active site structures

Conserved structures, except for active site loop:protein-protein interactions ?

2QJJ3BSM3DFH3GY1

Current directions

Unknown family in the MLE subgroup: 2PMQ

MLEMLEIIMLE 2OSBSNSAR

NSAR 2Dipeptide epimerase

Unknown

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

PGI PFKFBP Aldolase

TIM G3PDH PGK PGM Enolase

Rank (%) 0.9 0.1 0.3 0.1 0.8 0.5 0.03 0.2

PDBHM 64% to

2cxr1pfk

HM 40% to 3elf

7timHM 58% to 1nqa

HM 45% to 1vpe

HM 49% to 1ejj

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

2PMQ gene cluster(Pelagibaca bermudensis)

Transporter: A Trp “cage” for a betaine

Template: 1O7N smaller active site: indole in

1O7N has lots of steric clashes

Dioxygenase/hydroxylase: Homologues use aromatics

Closest liganded homologues: 60% with 3N0Q, unliganded18% with 1O7N: a naphthalene dioxygenase, cocrystalized with indole

1 2 3 4 5

Docked with 4-hydroxy proline

Top 5 docking hits

2PMQ: Docking, a small active site

4-OH Pro betaine

kcat/Km = 4300 M-1s-1

Experimental testing of 2PMQ prediction

H3C CH3

Pro betaine

kcat/Km = 380 M-1s-1

N+ CO2-

H3C CH3

N+ CO2-

H3C CH3

Genome context was helpful, but structures were essential

First amino acid racemase in EN superfamily

Proposed pathway for 4-OH Pro utilization

Metabolomics to confirm pathway is in progress

N+ CO2-

H3C CH3

N+ CO2-

H3C CH3

CO2--O2C

Current directions

SSN: dehydratases in EN superfamilyMandelate racemase

D-Glucarate dehydrataseD-Mannonate dehydratase

Galactarate dehydrataseD-Arabinonate dehydratase

Galactonate dehydrataseGluconate dehydrataseD-Tartrate dehydratase

L-Fuconate dehydrataseGalactarate/L-talarate dehydratase

L-Rhamnonate dehydratase

Unknown

Nine Agrobacterium tumefaciens dehydratasesFour SNFs

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

Library screening: 1RVK is a novel GlucD

D-Glucarate

L-Idarate

kcat = 0.36 s-1

Km = 45 μM

kcat/Km = 7.4 x 103 M-1 s-1

kcat = 0.33 s-1

Km = 54 μM

kcat/Km = 6.1 x 103 M-1 s-1

Agrobacterium utilize D-glucarate as carbon source ??

Complex with L-lyxarohydroxamate

Novel pathway for D-glucuronate catabolism ?

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

Current directions

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

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