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 superfamily: > 20 assigned functions
CO2OH
H
(R)-Mandelate
-O
OPO322C
CH2OH
H
2-PGA
CO2H
CH3
+H N
H
3
CO2-
-O
- H2O
anti
H
CO2-
PEP
-O
MAL
H CO2
CO2H
O C
2C
O
Ala
Muconolactone
CO2N
H H
-O
2C
CO2O
OSB
-O
2C
OH
NH
Ala
+H N
2
NSAR
CO2NH
H
N-Succ-L-Arg
CO2-
L-Ala-D-Glu
NH2
O
CO2-
- H2O
syn
AE Epim
HN
H
CO2-
syn
L-Ala-L-Glu
+H N
2
O
OSBS
O
SHCHC
-O
HH
H
CO2-
-
Mesaconate
MLE
-O
HN
2C
FucD
- H2O
CO2O
H H
HO H
H OH
CH2OH
CO2O
H H
H OH
HO H
CH2OH
CO2O
H H
HO H
HO H
CH3
CO2OH
H
OH
CH2OH
CO2-
CO2OH
H
OH
OH
CH2OH
D-Gluconate
H
HO
H
H
HO
H
CO2H
OH
CO2-
GlcD
- H2O
TarD
- H2O
CO2O
H H
H OH
H OH
CH2OH
CO2O
H H
CO2-
XylD
- H2O
CO2H
GlucD
H
OH
- H2O
H
syn
CO2D-Glucarate
HO
HO
H
HO
HO
H
H
CO2H
OH
OH
CH2OH
AraD
- H2O
CO2O
H H
H OH
CH2OH
D-Arabinonate
CO2CO2HO H GalrD/TalrD
O
H OH
H H
H OH
H OH
- H2O
HO H
HO H
CO2
CO2Galactarate
H
HO
H
CO2H
OH
(S)-Mandelate
D-Tartrate
CO2H OH
RhamD
H OH
HO H
- H2O
HO H
CH3
L-Rhamnonate
CO2O
H H
H OH
CH2OH
D-Xylonate
NH2
OH
NH
CO2H
OH
OH
H
CH3
L-Fuconate
HO
H
H
HO
CH3
2C
-NH3
anti
cis,cis-Muconate
HO H
2C
CH2
3-Methyl Asp
O- H
O C
OPO32-
Enolase
CO2OH
GalD
H
H
- H2O
OH
CH2OH
D-Galactonate
H
HO
HO
H
MR
CO2O
H H
H OH
HO H
CO2-
CO2CO2OH GalrD/TalrD
O
OH
H H
OH
H OH
- H2O
H
HO H
CO2
CO2L-Talarate
H
H
H
HO
CO2OH GlucD-II
H
OH
- H2O
OH
CO2D-Glucarate
H
HO
H
H
CO2OH
H
OH
H
CO2L-Idarate
H
HO
H
HO
GlucD
- H2O
anti
CO2O
H H
H OH
HO H
CO2CO2O
H H
H OH
HO H
CO2-
N-Succ-D-Arg
CO2OH
ManD
OH
OH
- H2O
OH
syn
CH2OH
D-Mannonate
H
H
H
H
CO2O
H H
H OH
H OH
CH2OH
CO2H
OH
OH
H
CO2Galactarate
HO
H
H
HO
GalrD
- H2O
anti
HO
H
H
CO2H
OH
H
O
CO2-
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 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
SSN: dehydratases in EN superfamily
D-Mannonate dehydratase
ManD
Boundaries between functions: ManD
e-80  35% seq.id.
e-184  70% seq.id.
≥ 70% sequence identity: functional significance ?
Activities: ManD, low ManD and/or GluD, none
Structures
Conserved active site structures
2QJJ
3BSM
3DFH
3GY1
Conserved structures, except for active site loop:
protein-protein interactions ?
2QJJ
3BSM
3DFH
3GY1
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
MLE
MLEII
MLE 2
OSBS
NSAR
NSAR 2
Dipeptide epimerase
Unknown
2PMQ: Structure with No Function (SNF) from PSI-2
Operon docking: retrospective glycolysis
C. Kalyanaraman
and M. P. Jacobson.
"Studying enzymesubstrate specificity
in silico: A case
study of the E. coli
glycolysis pathway”,
Biochemistry, 49
(2010) 4003-4005.
PGI
PFK
FBP
Aldolase
TIM
G3PDH
PGK
PGM
Enolase
Rank (%)
0.9
0.1
0.3
0.1
0.8
0.5
0.03
0.2
PDB
HM 64%
to 2cxr
1pfk
HM 40% to
HM 58% HM 45% HM 49%
7tim
3elf
to 1nqa to 1vpe to 1ejj
1ebj
2PMQ gene cluster
(Pelagibaca bermudensis)
Transporter: A Trp “cage” for a betaine
Dioxygenase/hydroxylase: Homologues use aromatics
Closest liganded homologues: 60% with 3N0Q, unliganded
18% with 1O7N: a naphthalene dioxygenase, cocrystalized with indole
Template: 1O7N
smaller active site: indole in
1O7N has lots of steric clashes
2PMQ: Docking, a small active site
1
2
3
4
5
Docked with 4-hydroxy
proline
Top 5 docking hits
Experimental testing of 2PMQ prediction
HO
HO
CO2-
N+
H3C
H3C
CH3
CO2-
N+
kcat/Km = 4300 M-1s-1
CH3
4-OH Pro betaine
CO2-
N+
H3C
CH3
CO2
N+
H3C
-
kcat/Km = 380 M-1s-1
CH3
Pro betaine
Genome context was helpful, but structures were essential
First amino acid racemase in EN superfamily
Proposed pathway for 4-OH Pro utilization
HO
HO
HO
2PMQ
N+
H3C
CO2-
N+
H3C
CH3
CO2-
CO2-
N
CH3
CH3
HO
HO
N
CO2-
N
CO2-
H2N
CO2-
HO H
O
H
H
CO2-
O
O
-O C
2
CO2O
Metabolomics to confirm pathway is in progress
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 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
Nine Agrobacterium tumefaciens dehydratases
Four SNFs
FucD
1RVK
3DIP
2NQL
3TJ4
1RVK: ordered 20s loop, large active site ?
Library screening: 1RVK is a novel GlucD
HO
HO
H
HO
CO2H
H
OH
H
CO2-
1RVK
- H2O
CO2O
H
H
H
OH
H
HO
CO2-
D-Glucarate
kcat = 0.36 s-1
Km = 45 M
kcat/Km = 7.4 x 103 M-1 s-1
H
HO
H
HO
CO2OH
H
OH
H
CO2-
1RVK
- H2O
CO2O
H
H
H
OH
H
HO
CO2-
L-Idarate
kcat = 0.33 s-1
Km = 54 M
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 ?
Phenotypic/metabolomic analyses by Micro Core
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
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