Cloned (Comment) | Organism |
---|---|
gene TRII, DNA and amino acid sequence determination and analysis, phylogenetic analysis and tree, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), realtime PCR enzyme expression analysis | Dendrobium nobile |
Protein Variants | Comment | Organism |
---|---|---|
Y201V | site-directed mutagenesis, the mutant enzyme shows restored activity with tropinone, while the wild-type is inactive with tropinone. The mutant enzymes also remain to have the ability to reduce the 3-quinuclidinone and 4-methylcyclohexanone, although the Vmax of DnTR2-Y201V toward 4-methylcyclohexanone and 3-quinuclidinone are significantly lower (23 and 3fold decrease, respectively) than that of wild-type DnTR2 protein | Dendrobium nobile |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
29420 | - |
- |
Dendrobium nobile |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Dendrobium nobile | H9BQR9 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Dendrobium nobile |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
leaf | - |
Dendrobium nobile | - |
additional information | TR2 shows constitutive expression in all tissues | Dendrobium nobile | - |
root | - |
Dendrobium nobile | - |
stem | - |
Dendrobium nobile | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
3-quinuclidinone + NADPH + H+ | wild-type and mutant Y201V enzymes | Dendrobium nobile | 1-azabicyclo[2.2.2]octan-3-ol + NADP+ | - |
r | |
4-methylcyclohexanone + NADPH + H+ | wild-type and mutant Y201V enzymes | Dendrobium nobile | 4-methylcyclohexanol + NADP+ | - |
r | |
tropinone + NADPH + H+ | recombinant enzyme mutant Y201V, no activity by wild-type TR2 enzyme | Dendrobium nobile | pseudotropine + NADP+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
? | x * 29420, sequence calculation | Dendrobium nobile |
More | three-dimensional structure modeling of DnTR2, structure comparisons, overview | Dendrobium nobile |
Synonyms | Comment | Organism |
---|---|---|
DnTR2 | - |
Dendrobium nobile |
TR2 | - |
Dendrobium nobile |
TRII | - |
Dendrobium nobile |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Dendrobium nobile |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
tropinone reduction assay at | Dendrobium nobile |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
additional information | the enzyme contains the conserved NAD(P)H binding motif (Gly-X3-Gly-X-Gly) | Dendrobium nobile | |
NADP+ | - |
Dendrobium nobile | |
NADPH | - |
Dendrobium nobile |
Organism | Comment | pI Value Maximum | pI Value |
---|---|---|---|
Dendrobium nobile | sequence calculation | - |
6.44 |
Organism | Comment | Expression |
---|---|---|
Dendrobium nobile | the enzyme expression is induced in leaves, stems, and rootsby methyljasmonate (highest in leaves, peaks after 24 h), and salicylate (peaks after 8 h), as well as by NO (peaks after 16 h) | up |
General Information | Comment | Organism |
---|---|---|
evolution | DnTR2 amino acid sequence contains a conserved Rossmann folding structure, which includes a conserved NAD(P)H binding motif (Gly-X3-Gly-X-Gly) and catalytic residues Ser-Asn-Lys, suggesting that DnTR2 is a member of the SDR superfamily | Dendrobium nobile |
malfunction | DnTR2 residue Tyr201 is located at the opposite side of Arg110 in the inner substrate binding surface. These structural characters suggest that the disabled tropinone reduction activity of DnTR2 may be caused by the replacement of an uncharged amino acid at position 201 | Dendrobium nobile |
additional information | three-dimensional structure modeling of DnTR2, catalytic triad Ser-Asn-Lys, role of Tyr201 in substrate binding, structure comparisons, overview | Dendrobium nobile |