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Literature summary for 1.1.1.206 extracted from

  • Kushwaha, A.K.; Sangwan, N.S.; Trivedi, P.K.; Negi, A.S.; Misra, L.; Sangwan, R.S.
    Tropine forming tropinone reductase gene from Withania somnifera (Ashwagandha): biochemical characteristics of the recombinant enzyme and novel physiological overtones of tissue-wide gene expression patterns (2013), PLoS ONE, 8, e74777.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene TR-I from roots, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis and tree, recombinant functional overexpression of His6-tagged enzyme in Escherichia coli strain BL21(DE3) Withania somnifera

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics Withania somnifera
0.00043
-
NADPH recombinant enzyme, pH 6.7, 30°C Withania somnifera
0.0962
-
NADP+ recombinant enzyme, pH 6.7, 30°C Withania somnifera
1.27
-
tropinone recombinant enzyme, pH 6.7, 30°C Withania somnifera
1.74
-
N-methylpiperidine recombinant enzyme, pH 6.7, 30°C Withania somnifera
2.11
-
3-methylcyclohexanone recombinant enzyme, pH 6.7, 30°C Withania somnifera
114.9
-
Tropine recombinant enzyme, pH 6.7, 30°C Withania somnifera

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ slightly activating Withania somnifera
additional information except slight enhancement of enzyme activity by Mg2+ and Na+, none of the following cations, Mn2+, Li+, NH4+, Mg2+, Na+, and Co2+, 5 mM each, affects the activity significantly Withania somnifera
Na+ slightly activating Withania somnifera

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
60000
-
2 * 60000, SDS-PAGE Withania somnifera

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
tropinone + NADPH + H+ Withania somnifera
-
tropine + NADP+
-
r

Organism

Organism UniProt Comment Textmining
Withania somnifera U5PUZ1 cv. NMITLI-118
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by affinity chromatography and gel filtration Withania somnifera

Source Tissue

Source Tissue Comment Organism Textmining
berry low enzyme level, all developmental stages Withania somnifera
-
leaf low enzyme level, all developmental stages Withania somnifera
-
additional information enzyme activity and tropine/tropinone contents during different developmental stages, overview Withania somnifera
-
root high enzyme level in bark, moderate in medulla Withania somnifera
-
shoot high enzyme level Withania somnifera
-
stem high enzyme level in bark, moderate in medulla Withania somnifera
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1-propyl-4-piperidone + NADPH + H+ low activity Withania somnifera 1-propylpiperidin-4-ol + NADP+
-
?
2-pyrrolidone + NADPH + H+ very low activity Withania somnifera 2-hydroxypyrrolidine + NADP+
-
?
3-methylcyclohexanone + NADPH + H+
-
Withania somnifera 3-methylcyclohexanol + NADP+
-
?
4-chloro-1-methylpiperidine + NADPH + H+ moderate activity Withania somnifera ? + NADP+
-
?
4-ethylcyclohexanone + NADPH + H+ low activity Withania somnifera 4-ethylcyclohexanol + NADP+
-
?
4-methylcyclohexanone + NADPH + H+ low activity Withania somnifera 4-methylcyclohexanol + NADP+
-
?
4-piperidone + NADPH + H+ very low activity Withania somnifera 4-hydroxypiperidin + NADP+
-
?
exo-6-hydroxytropinone + NADPH + H+ low activity Withania somnifera ? + NADP+
-
?
additional information TLC and GC-MS analysis of substrates and products. The enzyme also shows comparatively efficient catalysis of NADPH-dependent reduction of cyclic ketones 3-methylcyclohexanone and N-methylpiperidine, substrate specificity, overview. No activity with 1-methyl-2-pyrrolidone Withania somnifera ?
-
?
N-methyl-4-piperidone + NADPH + H+ low activity Withania somnifera 1-methylpiperidin-4-ol + NADP+
-
?
N-methylpiperidine + NADPH + H+
-
Withania somnifera ? + NADP+
-
?
tetrahydro-4H-thiopyran-4-one + NADPH + H+ moderate activity Withania somnifera ? + NADP+
-
?
tropine + NADP+ catalytic kinetics of the enzyme favour the forward reaction, tropine formation Withania somnifera tropinone + NADPH + H+
-
r
tropinone + NADPH + H+
-
Withania somnifera tropine + NADP+
-
r

Subunits

Subunits Comment Organism
homodimer 2 * 60000, SDS-PAGE Withania somnifera
More the homodimer is the catalytically active form of the recombinant WsTR-I Withania somnifera

Synonyms

Synonyms Comment Organism
TR-I
-
Withania somnifera
tropinone reductase
-
Withania somnifera
tropinone reductase-I
-
Withania somnifera
WsTR-I
-
Withania somnifera
WsTR-I protein
-
Withania somnifera

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Withania somnifera

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
50 60 purified recombinant enzyme, retains about 75% of maximal catalytic activity at up to 50°C, 4.27% activity remaining on incubation at 60°C Withania somnifera

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.54
-
NADP+ recombinant enzyme, pH 6.7, 30°C Withania somnifera
1.67
-
Tropine recombinant enzyme, pH 6.7, 30°C Withania somnifera
8.97
-
3-methylcyclohexanone recombinant enzyme, pH 6.7, 30°C Withania somnifera
9.23
-
N-methylpiperidine recombinant enzyme, pH 6.7, 30°C Withania somnifera
9.51
-
tropinone recombinant enzyme, pH 6.7, 30°C Withania somnifera
33.34
-
NADPH recombinant enzyme, pH 6.7, 30°C Withania somnifera

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.7
-
-
Withania somnifera

Cofactor

Cofactor Comment Organism Structure
NADP+
-
Withania somnifera
NADPH
-
Withania somnifera

Expression

Organism Comment Expression
Withania somnifera the enzyme-encoding TR-I gene expression is upregulated on exposure to signal molecules, i.e. methyl jasmonate and salicylic acid, and on mechanical injury/wounding up

General Information

General Information Comment Organism
evolution molecular evolution of WsTR-I, overview Withania somnifera
metabolism the enzyme catalyzes the NADPH-dependent tropinone to tropine conversion step in tropane metabolism, metabolic pathway of tropane alkaloid biosynthesis, overview Withania somnifera
additional information enzyme three-dimensional structure modelling Withania somnifera
physiological function physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The metabolic step of tropine formation may be regulated by, besides through the transcript and protein levels, the tissue concentration of tropinone Withania somnifera

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.014
-
Tropine recombinant enzyme, pH 6.7, 30°C Withania somnifera
0.559
-
NADP+ recombinant enzyme, pH 6.7, 30°C Withania somnifera
4.38
-
N-methylpiperidine recombinant enzyme, pH 6.7, 30°C Withania somnifera
5.17
-
3-methylcyclohexanone recombinant enzyme, pH 6.7, 30°C Withania somnifera
7.49
-
tropinone recombinant enzyme, pH 6.7, 30°C Withania somnifera
77540
-
NADPH recombinant enzyme, pH 6.7, 30°C Withania somnifera