Literature summary for 1.1.1.B52 extracted from

Hou, F.; Miyakawa, T.; Kataoka, M.; Takeshita, D.; Kumashiro, S.; Uzura, A.; Urano, N.; Nagata, K.; Shimizu, S.; Tanokura, M.
Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR (2014), Biochem. Biophys. Res. Commun., 446, 911-915 .

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Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of N-terminally His6-tagged enzyme in Eschrichia coli strain Rosetta(DE3)	Agrobacterium tumefaciens

Crystallization (Commentary)

Crystallization (Comment)	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

Crystallization (Comment)

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

Protein Variants

Protein Variants	Comment	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	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
3-quinuclidinone + NADH + H+	Agrobacterium tumefaciens	stereospecific reduction of 3-quinuclidinone	(R)-3-quinuclidinol + NAD+	-	?

Organism

Organism	UniProt	Comment	Textmining
Agrobacterium tumefaciens	G1K3P5	-	-

Purification (Commentary)

Purification (Comment)	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	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
3-quinuclidinone + NADH + H+	stereospecific reduction of 3-quinuclidinone	Agrobacterium tumefaciens	(R)-3-quinuclidinol + NAD+	-	?

Subunits

Subunits	Comment	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

Synonyms

Synonyms	Comment	Organism
3-quinuclidinone reductase	-	Agrobacterium tumefaciens
AtQR	-	Agrobacterium tumefaciens
NADHdependent 3-quinuclidionone reductase	-	Agrobacterium tumefaciens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Agrobacterium tumefaciens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Agrobacterium tumefaciens

Cofactor

Cofactor	Comment	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

General Information

General Information	Comment	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