Information on EC 2.1.1.160 - caffeine synthase

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The expected taxonomic range for this enzyme is: Gunneridae

EC NUMBER
COMMENTARY hide
2.1.1.160
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RECOMMENDED NAME
GeneOntology No.
caffeine synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + 1,7-dimethylxanthine = S-adenosyl-L-homocysteine + 1,3,7-trimethylxanthine
show the reaction diagram
Paraxanthine is the best substrate for this enzyme but the paraxanthine pathway is considered to be a minor pathway for caffeine biosynthesis.
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S-adenosyl-L-methionine + 3,7-dimethylxanthine = S-adenosyl-L-homocysteine + 1,3,7-trimethylxanthine
show the reaction diagram
S-adenosyl-L-methionine + 7-methylxanthine = S-adenosyl-L-homocysteine + 3,7-dimethylxanthine
show the reaction diagram
Paraxanthine is the best substrate for this enzyme but the paraxanthine pathway is considered to be a minor pathway for caffeine biosynthesis.
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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caffeine biosynthesis I
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caffeine biosynthesis II (via paraxanthine)
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Caffeine metabolism
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:3,7-dimethylxanthine N1-methyltransferase
Paraxanthine is the best substrate for this enzyme but the paraxanthine pathway is considered to be a minor pathway for caffeine biosynthesis [2,3].
CAS REGISTRY NUMBER
COMMENTARY hide
155215-94-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
isozyme 1
SwissProt
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
CCS1
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Manually annotated by BRENDA team
Coffea sp.
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Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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post-transcriptional silencing of caffeine synthase mRNA leads to reduced caffeine content in tea leaves
metabolism
additional information
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regulation of TCS gene expression
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + 1,7-dimethylxanthine
S-adenosyl-L-homocysteine + 1,3,7-trimethylxanthine
show the reaction diagram
S-adenosyl-L-methionine + 1-methylxanthine
S-adenosyl-L-homocysteine + 1,3-dimethylxanthine
show the reaction diagram
S-adenosyl-L-methionine + 3,7-dimethylxanthine
S-adenosyl-L-homocysteine + 1,3,7-trimethylxanthine
show the reaction diagram
S-adenosyl-L-methionine + 3-methylxanthine
S-adenosyl-L-homocysteine + 1,3-dimethylxanthine
show the reaction diagram
S-adenosyl-L-methionine + 7-methylxanthine
S-adenosyl-L-homocysteine + 3,7-dimethylxanthine
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
S-adenosyl-L-methionine + 1,7-dimethylxanthine
S-adenosyl-L-homocysteine + 1,3,7-trimethylxanthine
show the reaction diagram
S-adenosyl-L-methionine + 3,7-dimethylxanthine
S-adenosyl-L-homocysteine + 1,3,7-trimethylxanthine
show the reaction diagram
S-adenosyl-L-methionine + 7-methylxanthine
S-adenosyl-L-homocysteine + 3,7-dimethylxanthine
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCN
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74% inhibition at 100 mM, but 33% activation at 10 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-chloromercuribenzoate
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complete inhibition at 0.5 mM, 23% inhibition at 0.05 mM
azouracil
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18% inhibition at 0.1 mM, no inhibition at 1 mM
iodoacetate
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complete inhibition at 100 mM, 89% inhibition at 10 mM
KCN
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74% inhibition at 100 mM, but 33% activation at 10 mM
N-Methylmaleimide
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complete inhibition at 1 mM, no inhibition at 0.1 mM
NaN3
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22% inhibition at 10 mM, 16% at 1 mM
additional information
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no inhibition by EDTA at 0.5-5 mM
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
paraxanthine
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i.e. 1,7-dimethylxanthine, slight stimulation
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.973
paraxanthine
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recombinant DXMT1
0.01 - 0.153
S-adenosyl-L-methionine
0.157 - 0.2
Theobromine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.65
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purified enzyme
95
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purified enzyme
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
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assay at
8.3
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 4.9
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chromatofocusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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caffeine synthase expression as well as caffeine content is found to be higher in commercially utilized tissues like apical bud, 1st leaf, 2nd leaf, young stem, and is lower in old leaf during non-dormant compared to dormant growth phase
Manually annotated by BRENDA team
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TCS transcripts content remains constant throughout fl ower development
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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co-localization of xanthosine methyltransferase, 7-methylxanthine methyltransferase, and 3,7-dimethylxanthine methyltransferase
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
41000
gel filtration
43000
SDS-PAGE
61000
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gel filtration
67000
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about, gel filtration
80000
about, recombinant enzyme, gel filtration
81000
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recombinant isozyme DXMT1, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 40000, SDS-PAGE; x * 41300, calculated from amino acid sequence
monomer or dimer
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x * 41000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant wild-type and selenomethionine-labeled DXMT, 23-28% PEG 3350, 0.2 M LiCl, 0.1 M Tris-HCl, pH 8.5-8.7, 2 mM DTT, 1 mM S-adenosyl-L-cysteine, and 1 mM theobromine, 1-3 days, 20C, X-ray diffraction structure determination and analysis at 2.5-2.7 A resolution
purified recombinant wild-type and selenomethionine-labeled DXMT, 23-28% PEG 3350, 0.2 M LiCl, 0.1 M Tris-HCl, pH 8.5-8.7, 2 mM DTT, 2 mM S-adenosyl-L-cysteine, and 2 mM theobromine, 1-3 days, 20C, plate-like crystals, X-ray diffraction structure determination and analysis at 2.0-2.7 A resolution, molecular replacement
using Linbro plates and the conventional hanging-drop technique, in the presence of the demethylated cofactor S-adenosyl-L-cysteine or theobromine
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme 24fold from young leaves by 3 steps of ion exchange chromatography, and gel filtration
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native enzyme 523fold from young, developing leaves by ammonium sulfate fractionation, hydroxylapatite, anion exchange, and adenosine affinity chromatography, and gel filtration to homogeneity
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recombinant His-tagged DXMT1 from Escherichia coli strain BL21(DE3) to homogeneity by nickel affinity chromatography, cleavage of the His-tag with tobacco etch virus, TEV, protease, followed by gel filtration
using the His-tagged affinity chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Agrobacterium tumefaciens-mediated silencing of caffeine synthesis through root transformation in Camellia sinensis suign the RNAi construct, pFGC1008-CS, method, overview
DNA and amino acid sequence determination and analysis, expression in Escherichia coli as wild-type and as Trx-fusion protein
DNA and amino acid sequence determination and analysis, expression of wild-type and chimeric mutant enzymes in Escherichia coli strain BL21(DE3), phylogenetic analysis
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli Rosetta BL21 (DE3) and Saccharomyces cerevisiae INVSC1 cells
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expression analysis
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expression in Escherichia coli
expression in Escherichia coli as His-tagged fusion protein
expression of His-tagged DXMT1 in Escherichia coli strain BL21(DE3)
expression of wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3), sequence comparisonof caffeine synthetic enzymes from coffee
Coffea sp.
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gene CaMTL1, cDNA library construction, DNA and amino acid sequence determination and analysis, sequence comparison with other species
gene DXMT1, DNA and amino acid sequence determination and analysis, expression analysis
gene TCS1, cDNA and amino acid sequence determination and analysis, expression in Escherichia coli
isozyme 1, DNA and amino acid sequence determination and analysis, expression of wild-type and chimeric mutant enzymes in Escherichia coli strain BL21(DE3), phylogenetic analysis
isozyme DXMT1, DNA and amino acid sequence determination and analysis, phylogenetic analysis, expression of the GST-tagged enzyme in Escherichia coli Bl21(DE3)
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isozyme DXMT1, functional expression in transgenic Nicotiana tabacum plant leaves
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semiquantitative-PCR for caffeine synthase transcript expression analysis
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
amounts of transcripts are much higher in young leaf than in other parts of the plant. Larger amounts of transcripts are found in young calluses than in old tissues
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caffeine synthase expression as well as caffeine content is found to be higher in commercially utilized tissues like apical bud, 1st leaf, 2nd leaf, young stem, and is lower in old leaf during non-dormant compared to dormant growth phase. Among fruit parts, fruit coats have higher caffeine synthase expression, caffeine content, and allantoin content
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drought stress suppresses TCS gene expression in leaves
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environmental stresses other than heavy metal stress and plant hormone treatments have no effect on the expression of TCS gene
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naturally occuring low-caffeine fruits have a lower expression of the theobromine synthase and caffeine synthase genes and also contain an extra transcript of the caffeine synthase gene encoding a mutant form. The absence of caffeine in these mutants probably results from a combination of transcriptional regulation and the presence of mutations
TCS transcript increases slightly on supply of a nitrogen source
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the expression of the enzyme is greatly decreased to 50% in the leaves of grafted tea leaves
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
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expression of the caffeine biosynthesis enzymes in transgenic crop plants may protect against the crop damaging larvae of pests
biotechnology
large-scale production of transgenic enzyme-deficient Coffea arabica and Camellia sinensis plants are a practical possibilty for production of decaffeinated coffee or tea
food industry