Information on EC 1.1.1.236 - tropinone reductase II

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.1.1.236
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RECOMMENDED NAME
GeneOntology No.
tropinone reductase II
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
pseudotropine + NADP+ = tropinone + NADPH + H+
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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redox reaction
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
calystegine biosynthesis
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Tropane, piperidine and pyridine alkaloid biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
pseudotropine:NADP+ 3-oxidoreductase
This enzyme along with EC 1.1.1.206, tropine dehydrogenase, represents a branch point in tropane alkaloid metabolism [3]. Tropine (the product of EC 1.1.1.206) is incorporated into hyoscyamine and scopolamine whereas pseudotropine (the product of EC 1.1.1.236) is the first specific metabolite on the pathway to the calystegines [3]. Both enzymes are always found together in any given tropane-alkaloid-producing species, have a common substrate, tropinone, and are strictly stereospecific [2].
CAS REGISTRY NUMBER
COMMENTARY hide
136111-61-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
x aurea hybrid
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
Hyoscyamus bohemicus
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Manually annotated by BRENDA team
Hyoscyamus canariensis
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Manually annotated by BRENDA team
no activity in Brassica campestris
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-
Manually annotated by BRENDA team
no activity in Browallia americana
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-
-
Manually annotated by BRENDA team
no activity in Nicotiana tabacum
tobacco
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
Physalis edulis
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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
metabolism
physiological function
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pseudotropine forming tropinone reductase (TRII) catalyzes a tropinone reduction competing with TRI, EC 1.1.1.206. StTRII is the main enzyme catalyzing the synthesis of pseudotropine from tropinone. StTRII plays a role in calystegine formation in potato sprouts
additional information
three-dimensional structure modeling of DnTR2, catalytic triad Ser-Asn-Lys, role of Tyr201 in substrate binding, structure comparisons, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-methyl-cyclohexanone + NADPH
2-methylcyclohexanol + NADP+
show the reaction diagram
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-
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?
3-methyl-cyclohexanone + NADPH
3-methylcyclohexanol + NADP+
show the reaction diagram
-
-
-
-
?
3-methyl-cyclohexanone + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
3-quinuclidinone + NADPH + H+
1-azabicyclo[2.2.2]octan-3-ol + NADP+
show the reaction diagram
wild-type and mutant Y201V enzymes
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-
r
4-ethyl-cyclohexanone + NADPH
4-ethylcyclohexanol + NADP+
show the reaction diagram
4-ethyl-cyclohexanone + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
4-methyl-cyclohexanone + NADPH
4-methylcyclohexanol + NADP+
show the reaction diagram
4-methyl-cyclohexanone + NADPH
? + NADP+
show the reaction diagram
4-methylcyclohexanone + NADPH + H+
4-methylcyclohexanol + NADP+
show the reaction diagram
wild-type and mutant Y201V enzymes
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-
r
4-piperidone + NADPH
piperidine-4-ol + NADP+
show the reaction diagram
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-
-
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?
4-tetrahydro-thiopyranone + NADP+
? + NADP+
show the reaction diagram
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-
-
-
?
4-tetrahydro-thiopyranone + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
6-hydroxytropinone + NADPH
? + NADP+
show the reaction diagram
N-(2-fluoroethyl)nortropinone + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
N-ethylnortropinone + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
N-iso-propylnortropinone + NADPH
? + NADP+
show the reaction diagram
-
-
-
-
?
N-methyl-4-piperidinone + NADPH
? + NADP+
show the reaction diagram
N-methyl-4-piperidone + NADPH
N-methylpiperidin-4-ol + NADP+
show the reaction diagram
N-methyltropinone + NADPH
N-methyl-3-beta-tropine + NADP+
show the reaction diagram
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-
-
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ir
N-propyl-4-piperidinone + NADPH
? + NADP+
show the reaction diagram
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-
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?
N-propyl-4-piperidone + NADPH
N-propylpiperidin-4-ol + NADP+
show the reaction diagram
nortropinone + NADPH
? + NADP+
show the reaction diagram
psi-tropine + NADP+
tropinone + NADPH
show the reaction diagram
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r
tetrahydrothiopyran-4-one + NADPH
tetrahydrothiopyran-4-ol + NADP+
show the reaction diagram
tropine + NADP+
tropinone + NADPH
show the reaction diagram
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r
tropinone + NADPH + H+
pseudotropine + NADP+
show the reaction diagram
tropinone + NADPH + H+
tropine + NADP+
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
tropinone + NADPH + H+
pseudotropine + NADP+
show the reaction diagram
tropinone + NADPH + H+
tropine + NADP+
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
NADPH
additional information
the enzyme contains the conserved NAD(P)H binding motif (Gly-X3-Gly-X-Gly)
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
methyl jasmonate
inhibits expression of TRII in hairy roots
p-chloromercuribenzoic acid
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
methyl jasmonate can enhance expression of TRII, with strong effect for TRII. TRII transcript is strongly induced, reaching the highest level after 24 h of the treatment in root and stem. TRII transcription level is highest in leaf after 12h treatment
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7.58
3-methyl-cyclohexanone
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7.58
3-methylcyclohexanone
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0.534
4-ethyl-cyclohexanone
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0.534
4-ethylcyclohexanone
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2.03 - 2.8
4-methyl-cyclohexanone
2.03 - 2.8
4-methylcyclohexanone
21.6
4-Piperidone
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0.38 - 2
4-tetrahydro-thiopyranone
0.67
N-(2-fluoroethyl)nortropinone
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0.25
N-ethylnortropinone
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0.65 - 20
N-methyl-4-piperidinone
0.057 - 0.77
N-Methyl-4-piperidone
1.4
N-methylpiperid-4-one
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0.265
N-propyl-4-piperidinone
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0.265 - 0.299
N-propylyl-4-piperidone
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0.0076
NADH
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0.251
NADP+
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0.00172 - 0.0255
NADPH
0.025
Nortropinone
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0.033 - 52.5
tropinone
additional information
additional information
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the recombinant His-tagged enzyme shows an increased substrate affinity compared to the wild-type enzyme
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
71.4
NADPH
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pH 7.5, 15C
2.73 - 71.4
tropinone
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0145
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additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 5
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optimum for tetrahydrothiopyran-4-one reduction
5.3 - 6.5
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6.8
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tropinone reduction
8
tropinone reduction assay at
9.5
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tropine oxidation
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 5
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reverse oxidative activities
4.5 - 8.5
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tolerant to pH changes over a wide range
additional information
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the recombinant His-tagged enzyme shows a broader pH spectrum compared to the wild-type enzyme
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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tropine oxidation
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.35
sequence analysis
6.44
sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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root culture
Manually annotated by BRENDA team
additional information
TR2 shows constitutive expression in all tissues
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25000 - 55000
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TRII preparation is not yet pure and shows numerous bands
27500
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3 * 27500
27700
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3 * 27700, SDS-PAGE
28290
sequence analysis
28400
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SDS-PAGE
39000
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2 * 39000, SDS-PAGE
69000
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minor peak, gel filtration
77700
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gel filtration
84000
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gel filtration
103000
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major peak, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
trimer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
TrII is crystallized by hanging-drop vapor diffusion with 2-methyl-2,4-pentanediol as precipitating reagent, crystals grown by macroseeding technique, crystals belong to the P4.2.2.1.2 space group with cell dimensions of a = b = 62.8 A and c = 128.4 A
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 8
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keeps 90% of maximal activity in the range pH 5.2-8.2
348027
5.8 - 8.5
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more acidic buffers inactivate the enzyme highly, 8% of the maximum activity at pH 4.5, more alkaline buffers inhibit the activity less strongly, 61% of the maximum activity at pH 8.5
348021, 348025
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24 - 56
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77% of the maximum activity at 24C, completely inactivated at 56C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
activity rapidly lost during purification
denatured enzyme does not catalyze the reduction of tropinone with NADPH
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very unstable
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, enzyme preparation can be stored for 3 months without loss of activity
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-20C, stable for at least 3 months
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4C, enzyme preparation can be stored for 4 days without loss of activity, when kept in 10% glycerol enzyme activity is lost within 2 days
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
HiTrap chelating HP Sepharose column chromatography
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant StTRII L1
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cDNA coding for TRI and TRII isolated and expressed in Escherichia coli NM522, P29X-encoding gene detected
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cDNA encoding tropinone reductase II
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cDNA homologous to pseudotropine-forming tropinone reductase isolated, recombinant protein functionally expressed in Escherichia coli BL21(DE3), transformed with pET21d vectors
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expressed in Escherichia coli
full-length sequences determined of 5 clones with high homology to TRs
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gene tr, DNA and amino acid sequence determination and analysis, sequence comparison
gene TR2, enzyme overexpression via leaves transformation with Agrobacterium tumefaciens AGL0, quantitative PCR enzyme expression analysis
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gene trII, DNA and amino acid sequence determination and analysis, expression as C- or N-terminally His-tagged enzyme in Escherichia coli strain SG13009, the His-tage posotion influences the pH dependence of the enzyme, oveview
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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
overexpression in Atropa belladonna. Transformation with cDNA of tropinone reductases successfully alters the ratio of tropine-derived alkaloids versus pseudotropine-derived alkaloids
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P29X-encoding gene detected
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TRII cDNA subcloned from pTR2EN induced in Escherichia coli BL21(DE3)
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TRII cDNA, in Escherichia coli expression vector pET21d
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
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)
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
additional information
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enzyme suppression in potato plants by RNA interference (RNAi) and enzyme overexpression. The overexpression and gene knockdown of StTRII alters tropinone reduction in vivo, and has great influence on calystegine accumulation, in contrast to genetic manipulation of isozyme St-TRI, EC 1.1.1.206, phenotypes, calystegine analysis, overview. A strong reduction of calystegines appears in the StTRII RNAi lines