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Literature summary 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 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.B52 recombinant expression of N-terminally His6-tagged enzyme in Eschrichia coli strain Rosetta(DE3) Agrobacterium tumefaciens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.B52 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

EC Number Protein Variants Comment Organism
1.1.1.B52 D40A site-directed mutagenesis, inactive mutant Agrobacterium tumefaciens
1.1.1.B52 E197A site-directed mutagenesis, inactive mutant Agrobacterium tumefaciens
1.1.1.B52 R196A site-directed mutagenesis, the mutant shows 34% reduced activity compared to the wild-type enzyme Agrobacterium tumefaciens
1.1.1.B52 Y216V site-directed mutagenesis, the mutant shows 69% reduced activity compared to the wild-type enzyme Agrobacterium tumefaciens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.B52 3-quinuclidinone + NADH + H+ Agrobacterium tumefaciens stereospecific reduction of 3-quinuclidinone (R)-3-quinuclidinol + NAD+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.B52 Agrobacterium tumefaciens G1K3P5
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.B52 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.B52 3-quinuclidinone + NADH + H+ stereospecific reduction of 3-quinuclidinone Agrobacterium tumefaciens (R)-3-quinuclidinol + NAD+
-
 ?

Subunits

EC Number Subunits Comment Organism
1.1.1.B52 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

EC Number Synonyms Comment Organism
1.1.1.B52 3-quinuclidinone reductase
-
 Agrobacterium tumefaciens
1.1.1.B52 AtQR
-
 Agrobacterium tumefaciens
1.1.1.B52 NADHdependent 3-quinuclidionone reductase
-
 Agrobacterium tumefaciens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.B52 37
-
 assay at Agrobacterium tumefaciens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.B52 7
-
 assay at Agrobacterium tumefaciens

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.B52 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

EC Number General Information Comment Organism
1.1.1.B52 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
1.1.1.B52 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