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