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show all sequences of 1.1.1.B52

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

Hou, F.; Miyakawa, T.; Kataoka, M.; Takeshita, D.; Kumashiro, S.; Uzura, A.; Urano, N.; Nagata, K.; Shimizu, S.; Tanokura, M.; Biochem. Biophys. Res. Commun. 446, 911-915 (2014)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
recombinant expression of N-terminally His6-tagged enzyme in Eschrichia coli strain Rosetta(DE3)
Agrobacterium tumefaciens
Crystallization (Commentary)
Crystallization
Organism
purified recombinant His-tagged enzyme, enzyme AtQR and 2 mM NADH are crystallized from a reservoir solution containing of 0.2 M ammonium acetate, 0.1 M HEPES, pH 8.5, and 24% w/v PEG 3350, X-ray diffraction structure determination and analysis at 1.72 A resolution. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the alpha7 helix. Molecular replacement using structure of meso-2,3-butanediol dehydrogenase, PDB ID 1GEG, from Klebsiella pneumoniae as template
Agrobacterium tumefaciens
Engineering
Amino acid exchange
Commentary
Organism
D40A
site-directed mutagenesis, inactive mutant
Agrobacterium tumefaciens
E197A
site-directed mutagenesis, inactive mutant
Agrobacterium tumefaciens
R196A
site-directed mutagenesis, the mutant shows 34% reduced activity compared to the wild-type enzyme
Agrobacterium tumefaciens
Y216V
site-directed mutagenesis, the mutant shows 69% reduced activity compared to the wild-type enzyme
Agrobacterium tumefaciens
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
3-quinuclidinone + NADH + H+
Agrobacterium tumefaciens
stereospecific reduction of 3-quinuclidinone
(R)-3-quinuclidinol + NAD+
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Agrobacterium tumefaciens
G1K3P5
-
-
Purification (Commentary)
Commentary
Organism
recombinant N-terminally His6-tagged enzyme from Eschrichia coli strain Rosetta(DE3) by nickel affinity and anion exchange chromatography, and gel filtration
Agrobacterium tumefaciens
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3-quinuclidinone + NADH + H+
stereospecific reduction of 3-quinuclidinone
741816
Agrobacterium tumefaciens
(R)-3-quinuclidinol + NAD+
-
-
-
?
Subunits
Subunits
Commentary
Organism
tetramer
three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals, quaternary structure of AtQR, overview
Agrobacterium tumefaciens
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
37
-
assay at
Agrobacterium tumefaciens
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Agrobacterium tumefaciens
Cofactor
Cofactor
Commentary
Organism
Structure
NADH
three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the alpha7 helix. NADH is located in a deep cleft of the large domain and bound at the C-terminal end of the beta-sheet. The adenosine moiety of NADH is bound to a pocket formed by Gly16, Leu41, Val62, Asp63, Val64, Thr65, Ala91, Val93, and Val113. Residue Asp40 plays an important role in binding to NADH
Agrobacterium tumefaciens
Cloned(Commentary) (protein specific)
Commentary
Organism
recombinant expression of N-terminally His6-tagged enzyme in Eschrichia coli strain Rosetta(DE3)
Agrobacterium tumefaciens
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NADH
three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the alpha7 helix. NADH is located in a deep cleft of the large domain and bound at the C-terminal end of the beta-sheet. The adenosine moiety of NADH is bound to a pocket formed by Gly16, Leu41, Val62, Asp63, Val64, Thr65, Ala91, Val93, and Val113. Residue Asp40 plays an important role in binding to NADH
Agrobacterium tumefaciens
Crystallization (Commentary) (protein specific)
Crystallization
Organism
purified recombinant His-tagged enzyme, enzyme AtQR and 2 mM NADH are crystallized from a reservoir solution containing of 0.2 M ammonium acetate, 0.1 M HEPES, pH 8.5, and 24% w/v PEG 3350, X-ray diffraction structure determination and analysis at 1.72 A resolution. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the alpha7 helix. Molecular replacement using structure of meso-2,3-butanediol dehydrogenase, PDB ID 1GEG, from Klebsiella pneumoniae as template
Agrobacterium tumefaciens
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
D40A
site-directed mutagenesis, inactive mutant
Agrobacterium tumefaciens
E197A
site-directed mutagenesis, inactive mutant
Agrobacterium tumefaciens
R196A
site-directed mutagenesis, the mutant shows 34% reduced activity compared to the wild-type enzyme
Agrobacterium tumefaciens
Y216V
site-directed mutagenesis, the mutant shows 69% reduced activity compared to the wild-type enzyme
Agrobacterium tumefaciens
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
3-quinuclidinone + NADH + H+
Agrobacterium tumefaciens
stereospecific reduction of 3-quinuclidinone
(R)-3-quinuclidinol + NAD+
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant N-terminally His6-tagged enzyme from Eschrichia coli strain Rosetta(DE3) by nickel affinity and anion exchange chromatography, and gel filtration
Agrobacterium tumefaciens
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3-quinuclidinone + NADH + H+
stereospecific reduction of 3-quinuclidinone
741816
Agrobacterium tumefaciens
(R)-3-quinuclidinol + NAD+
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
tetramer
three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals, quaternary structure of AtQR, overview
Agrobacterium tumefaciens
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
37
-
assay at
Agrobacterium tumefaciens
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
assay at
Agrobacterium tumefaciens
General Information
General Information
Commentary
Organism
evolution
AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone
Agrobacterium tumefaciens
additional information
the alpha7 helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity, it is stabilized by NADH. An additional residue on the a7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Glu197 is an indispensable residue for the enzyme activity. Asp40 plays an important role in binding to NADH. Glu197 may be the key residue for enhancing the substrate-binding affinity. Structure-function anaysis and enantioselectivity, overview.
Agrobacterium tumefaciens
General Information (protein specific)
General Information
Commentary
Organism
evolution
AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone
Agrobacterium tumefaciens
additional information
the alpha7 helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity, it is stabilized by NADH. An additional residue on the a7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Glu197 is an indispensable residue for the enzyme activity. Asp40 plays an important role in binding to NADH. Glu197 may be the key residue for enhancing the substrate-binding affinity. Structure-function anaysis and enantioselectivity, overview.
Agrobacterium tumefaciens
Other publictions for EC 1.1.1.B52
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
729146
Hou
Structural basis for high subs ...
Agrobacterium tumefaciens
Biochem. Biophys. Res. Commun.
446
911-915
2014
-
2
1
1
4
1
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1
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738052
Spickermann
Engineering of highly selectiv ...
Parvibaculum lavamentivorans
ChemBioChem
15
2050-2052
2014
-
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1
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11
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1
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11
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2
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741816
Hou
Structural basis for high sub ...
Agrobacterium tumefaciens
Biochem. Biophys. Res. Commun.
446
911-915
2014
-
-
1
1
4
-
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1
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3
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1
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4
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1
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1
1
1
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1
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2
2
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730545
Zhang
Highly efficient synthesis of ...
Agrobacterium tumefaciens
Org. Lett.
15
4917-4919
2013
-
2
1
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1
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1
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1
1
741676
Isotani
Gene cloning and characteriza ...
Microbacterium luteolum, Microbacterium luteolum JCM 9174
Appl. Environ. Microbiol.
79
1378-1384
2013
-
1
1
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4
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4
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1
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11
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1
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2
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1
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1
1
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4
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1
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11
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1
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2
2
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1
1
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728893
Hou
Expression, purification, crys ...
Agrobacterium tumefaciens
Acta Crystallogr. Sect. F
68
1237-1239
2012
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1
1
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3
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729032
Isotani
Gene cloning and characterizat ...
Microbacterium luteolum, Microbacterium luteolum JCM 9174
Appl. Environ. Microbiol.
79
1378-1384
2012
-
2
1
-
-
-
-
4
-
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4
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4
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1
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2
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8
2
1
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2
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1
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1
1
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1
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2
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8
2
1
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2
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729854
Isotani
Production of (R)-3-quinuclidi ...
Microbacterium luteolum, Microbacterium luteolum JCM 9174
Int. J. Mol. Sci.
13
13542-13553
2012
-
2
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9
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2
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