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

2-

CH2OHH

-O2COPO3

2-

CH2

CO2-

CO2-

HOH

HOHO

HOHHH

syn

D-Glucarate

GlucD

CO2-

COO-

CO2-

COO H

HHH

H

cis,cis-Muconate Muconolactone

HO H

O

CO2-

CO2-

H

O

CO2-

CO2-

H

OSBSHCHC

CO2-

NH HAla

O

-O2C

CO2-

H

-O2C

NHAla

O

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

CO2-

CO2-

HOHH

HOHH

MLE

AE Epim

OSBS

anti

Enolase

O

2-PGA PEP

CO2-

CH2OH

OHOHOHOH

HHHH

CO2-

CH2OH

OHOHOH

HHH

D-Mannonate

ManD

syn

syn

syn

CO2-

CO2-

HOH

HOH

HOHHOH

L-Idarate

GlucD

CO2-

CO2-

HOHH

HOHHO

antiCO2

-

NHH

O CO2-

HNH

O

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

NSAR

-O2C-O2C

CO2-

CO2-

CH3H

H+H3N

3-Methyl Asp

CH3-O2C

CO2-H

Mesaconate

MAL

anti-NH3

HN HN

- H2O

- H2O

- H2O

- H2O

- H2O

CO2-

CO2-

HOHH

HO

HOHOHH

anti

Galactarate

GalrD

CO2-

CO2-

HH

HOHH

- H2O

+H2NNH2 +H2N

NH2

O

HO

CO2-

OHH

CO2-

HOH

(R)-Mandelate (S)-Mandelate

MR

CO2-

CH2OH

OHHHOH

HHOHO

H

CO2-

CH2OH

OHHOH

HHO

H

D-Galactonate

GalD

CO2-

CH3

OHOHHH

HH

HOHO

CO2-

CH3

OHHH

HHOHO

L-Rhamnonate

RhamD

CO2-

CH2OH

OHHOHOH

HHO

HH

CO2-

CH2OH

OHOHOH

HHH

D-Gluconate

GlcD

CO2-

CH3

HOHOHH

HOHH

HO

CO2-

CH2OH

OHOHH

HH

HO

L-Fuconate

FucD

CO2-

CO2-

HOH

HOH

CO2-

CO2-

OHH

D-Tartrate

TarD

CO2-

CO2-

HOHH

HO

HOHOHH

Galactarate

GalrD/TalrD

CO2-

CO2-

HOHH

HOHHO

CO2-

CO2-

HOHHH

HOHOHOH

L-Talarate

GalrD/TalrD

CO2-

CO2-

HOHH

HOHHO

- H2O - H2O

- H2O

- H2O - H2O

- H2O - H2O

CO2-

CH2OH

HOHOH

HOHH

CO2-

CH2OH

OHOH

HH

D-Arabinonate

AraD

- H2O

CO2-

CH2OH

OHHOH

HHO

H

CO2-

CH2OH

OHOH

HH

D-Xylonate

- H2O

XylD

CO2-

CO2-

OHHOHOH

HHO

HH

D-Glucarate

GlucD-II

CO2-

CO2-

HOHH

HOHHO

- H2O

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

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

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

ManD

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

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

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

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

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

N+CO2

-

HO

H3C CH3

N+CO2

-

HO

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-

HO

H3C CH3

N+ CO2-

HO

H3C CH3

NCO2

-

HO

CH3

NCO2

-

HO

H

NCO2

-

HO

O

CO2-

H2N

HO H

O

CO2-

O

H

O

CO2--O2C

2PMQ

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

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

FucD

3DIP

2NQL

1RVK

3TJ4

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

Library screening: 1RVK is a novel GlucD

CO2-

CO2-

HO

H

HO

HO

H

OH

H

H

D-Glucarate

1RVK

CO2-

CO2-

HO

H

H

H

OH

H

O

CO2-

CO2-

HO

H

HO

H

H

OH

H

OH

L-Idarate

CO2-

CO2-

HO

H

H

H

OH

H

O

- H2O

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

1RVK

- H2O

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

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