Information on EC 1.1.1.B52 - 3-quinuclidinone reductase (NADH)

for references in articles please use BRENDA:EC1.1.1.B52
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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
1.1.1.B52
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
3-quinuclidinone reductase (NADH)
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(R)-3-quinuclidinol + NAD+ = 3-quinuclidinone + NADH + H+
show the reaction diagram
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SYSTEMATIC NAME
IUBMB Comments
(R)-3-quinuclidinol:NAD+ oxidoreductase
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
additional information
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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.
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-acetylpyridine + NADH + H+
?
show the reaction diagram
2-acetylpyridine + NADH + H+
? + NAD+
show the reaction diagram
3-quinuclidinone + NADH + H+
(R)-3-quinuclidinol + NAD+
show the reaction diagram
7-oxabicyclo[4.1.0]heptan-2-one + NADH + H+
?
show the reaction diagram
7-oxabicyclo[4.1.0]heptan-2-one + NADH + H+
? + NAD+
show the reaction diagram
27.8% of the activity with 3-quinuclidinone
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
3-quinuclidinone + NADH + H+
(R)-3-quinuclidinol + NAD+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.4 - 13.8
3-quinuclidinone
0.02 - 0.03
NADH
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
290
3-quinuclidinone
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pH 7.0, temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.5
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pH 7.0, 25C, 3-quinuclidinone reductase BacC
8.4
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pH 7.0, 25C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8.5
activity range, profile overview
5.5 - 9.5
activity range, profile overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30600
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3 * 30600, SDS-PAGE, 3-quinuclidinone reductase QNR
32600
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2 * 32600, SDS-PAGE, 3-quinuclidinone reductase BacC
72000
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gel filtration, 3-quinuclidinone reductase BacC
94000
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gel filtration, 3-quinuclidinone reductase QNR
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
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three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals, quaternary structure of AtQR, overview
trimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystallized under a reservoir solution condition of 0.2 M ammonium acetate, 0.1 M HEPES pH 8.5 and 24% (w/v) PEG3350
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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
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sitting-drop vapour-diffusion method at 20C. Crystals are obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data are collected to 1.72 A resolution. The crystal belongs to space group P2(1), with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 A, beta = 110.5, and is suggested to contain four molecules in the asymmetric unit
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
NADH contributes to the stability of the alpha7 helix of the enzyme
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; 3-quinuclidinone reductase QNR
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native enzyme by anion exchange chromatography, dialysis, hydrophobic interaction chromatography, and dialysis, recombinant His-tagged enzyme from Escherichia coli 1.7fold by nickel affinity chromatography and ultrafiltration; native enzyme by anion exchange chromatography, dialysis, hydrophobic interaction chromatography, and dialysis, recombinant His-tagged enzyme from Escherichia coli 2.4fold by nickel affinity chromatography and ultrafiltration
recombinant N-terminally His6-tagged enzyme from Eschrichia coli strain Rosetta(DE3) by nickel affinity and anion exchange chromatography, and gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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expression in Escherichia coli. The gene is fused with the His6 tag and thrombin recognition sequence at the N-terminus under the control of the T7 promoter, 3-quinuclidinone reductase BacC; expression in Escherichia coli. The gene is fused with the His6 tag and thrombin recognition sequence at the N-terminus under the control of the T7 promoter, 3-quinuclidinone reductase QNR
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gene bacC, library construction, DNA and amino acid sequence determination and analysis, recombinant expression of His-tagged enzyme in Escherichia coli; gene qnr, library construction, DNA and amino acid sequence determination and analysis, recombinant expression of His-tagged enzyme in Escherichia coli
overexpressed as a fusion protein with an N-terminal His6 tag in Eschrichia coli Rosetta(DE3)
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overexpressed in Escherichia coli
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recombinant expression of N-terminally His6-tagged enzyme in Eschrichia coli strain Rosetta(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A158A/N162P
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96.8% enantiomeric excess for (R)-3-quinuclidinol production, 82% conversion
A158D/N162G
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96.5% enantiomeric excess for (R)-3-quinuclidinol production, 87% conversion
A158D/N162Q
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95.8% enantiomeric excess for (R)-3-quinuclidinol production, 79% conversion
A158H/N162G/K202Q/L224W
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mutations increase the enantiomeric excess for (R)-3-quinuclidinol production from 84.3% (wild-type) to 99% and concomitantly to enhance conversion by 43.5%
A158H/N162P
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98.1% enantiomeric excess for (R)-3-quinuclidinol production, 95% conversion
A158K/N162M
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97.6% enantiomeric excess for (R)-3-quinuclidinol production, 98% conversion
K202N/L224M
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89.7% enantiomeric excess for (R)-3-quinuclidinol production, 90% conversion
K202Q/L224W
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95.5% enantiomeric excess for (R)-3-quinuclidinol production, 82% conversion
K202R/L224W
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94.7% enantiomeric excess for (R)-3-quinuclidinol production, 91% conversion
K202S/L224Y
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89.5% enantiomeric excess for (R)-3-quinuclidinol production, 82% conversion
N162A
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93.8% enantiomeric excess for (R)-3-quinuclidinol production, 91% conversion
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pharmacology
synthesis