Cloned (Comment) | Organism |
---|---|
recombinant expression of N-terminally His6-tagged enzyme in Eschrichia coli strain Rosetta(DE3) | 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 | 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Agrobacterium tumefaciens | G1K3P5 | - |
- |
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
3-quinuclidinone + NADH + H+ | stereospecific reduction of 3-quinuclidinone | Agrobacterium tumefaciens | (R)-3-quinuclidinol + NAD+ | - |
? |
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 | Comment | Organism |
---|---|---|
3-quinuclidinone reductase | - |
Agrobacterium tumefaciens |
AtQR | - |
Agrobacterium tumefaciens |
NADHdependent 3-quinuclidionone reductase | - |
Agrobacterium tumefaciens |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Agrobacterium tumefaciens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Agrobacterium tumefaciens |
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 | 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 |