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Information on EC 2.1.1.292 - carminomycin 4-O-methyltransferase
for references in articles please use BRENDA:EC2.1.1.292
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EC Tree 2 Transferases
2.1 Transferring one-carbon groups
2.1.1 Methyltransferases
2.1.1.292 carminomycin 4-O-methyltransferase
IUBMB Comments
The enzymes from the Gram-positive bacteria Streptomyces sp. C5 and Streptomyces peucetius are involved in the biosynthesis of the anthracycline daunorubicin. In vitro the enzyme from Streptomyces sp. C5 also catalyses the 4-O-methylation of 13-dihydrocarminomycin, rhodomycin D and 10-carboxy-13-deoxycarminomycin .
The enzyme appears in viruses and cellular organisms
- 2.1.1.292
- methyltransferases
-
sam-dependent
- s-adenosyl-l-homocysteine
- daunorubicin
- peucetius
-
s-adenosyl-l-methionine-dependent
- synthesis
Reaction Schemes
showSynonyms
4-o-methyltransferase, class i methyltransferase, carminomycin 4-o-methyltransferase, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
DnrK
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + carminomycin = S-adenosyl-L-homocysteine + daunorubicin
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
BRENDA
MetaCyc
daunorubicin biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:carminomycin 4-O-methyltransferase
The enzymes from the Gram-positive bacteria Streptomyces sp. C5 and Streptomyces peucetius are involved in the biosynthesis of the anthracycline daunorubicin. In vitro the enzyme from Streptomyces sp. C5 also catalyses the 4-O-methylation of 13-dihydrocarminomycin, rhodomycin D and 10-carboxy-13-deoxycarminomycin [3].
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + (2S)-hesperetin
?
-
Substrates: 67% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + (2S)-naringenin
?
-
Substrates: 45% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 10-carboxy-13-deoxycarminomycin
S-adenosyl-L-homocysteine + 10-carboxy-13-deoxydaunorubicin
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + 11-deoxy-beta-rhodomycin T
S-adenosyl-L-homocysteine + 4-O-methyl-11-deoxy-beta-rhodomycin T
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + 13-deoxycarminomycin
S-adenosyl-L-homocysteine + 13-deoxydaunorubicin
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + 13-dihydrocarminomycin
S-adenosyl-L-homocysteine + 13-dihydrodaunomycin
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + 13-dihydrocarminomycin
S-adenosyl-L-homocysteine + daunorubicinol
S-adenosyl-L-methionine + 15-demethoxy-aclacinomycin T
S-adenosyl-L-homocysteine + 4-O-methyl-15-decarboxyaclacinomycin T
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + 2,4-dihydroxyquinoline
?
-
Substrates: 37% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 2-chloro-4-nitrophenol
?
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + 2-chloro-4-nitrophenol
S-adenosyl-L-homocysteine + methyl-2-chloro-4-nitrophenol
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + 4-hydroxyquinolin-2(1H)-one
?
-
Substrates: 51% conversion
Products: -
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?
S-adenosyl-L-methionine + 4-methylumbelliferone
S-adenosyl-L-homocysteine + 7-methoxy-4-methylcoumarin
-
Substrates: 12% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 5,4'-dimethoxy-7-hydroxyflavone
?
-
Substrates: 10% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 5,4'-dimethoxy-7-hydroxyisoflavone
S-adenosyl-L-homocysteine + 5,7,4'-trimethoxyisoflavone
-
Substrates: 18% conversion
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?
S-adenosyl-L-methionine + 5,7-dihydroxy-4-methylcoumarin
?
-
Substrates: 14% conversion
Products: -
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?
S-adenosyl-L-methionine + 5,7-dihydroxychrysin
?
-
Substrates: 26% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 5-carbomethoxymethyl-2-heptyl-7-hydroxychrome
?
-
Substrates: 98% conversion
Products: -
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?
S-adenosyl-L-methionine + 5-hydroxy-2',4',7,8-tetramethoxyflavone
?
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + 6,7-dihydroxycoumarin
?
-
Substrates: 44% conversion
Products: -
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?
S-adenosyl-L-methionine + 6,8-dihydroxy-7-methoxy-3-hydroxymethylisocoumarin
?
-
Substrates: 85% conversion
Products: -
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?
S-adenosyl-L-methionine + 6,8-dihydroxy-7-methoxy-3-methylisocoumarin
S-adenosyl-L-homocysteine + 8-hydroxy-6,7-dimethoxy-3-methylisocoumarin
-
Substrates: 56% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 6-hydroxy-7,8-dimethoxy-3-methylisocoumarin
?
-
Substrates: 65% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 7,4'-dihydroxyflavone
?
-
Substrates: 30% conversion
Products: -
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?
S-adenosyl-L-methionine + 7-deoxyaranciamycin
?
-
Substrates: also known as SM 173B, 96% conversion
Products: -
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?
S-adenosyl-L-methionine + 7-deoxynogalamycinone
?
-
Substrates: 62% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 7-hydroxyflavone
?
-
Substrates: 12% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 8-desmethyltetracenomycin C
?
-
Substrates: 85% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 8-hydroxy-3,4-dihydro-2-quinolinone
?
-
Substrates: 42% conversion
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?
S-adenosyl-L-methionine + 8-O-desmethylseitomycin
?
-
Substrates: 27% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 8-O-methylrabelomycin
?
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + 9,10-seco-7-deoxynogalamycinone
?
-
Substrates: 91% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + 9-methoxyalternariol
?
-
Substrates: 10% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + aclacinomycin T
S-adenosyl-L-homocysteine + triglycosylated aclacinomycin A
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + alizarin
?
-
Substrates: 13% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + apigenin dimethyl ether
?
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + aranciamycin
?
-
Substrates: 97% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + auramycinone
?
-
Substrates: 96% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + biochanin A
S-adenosyl-L-homocysteine + 7-methoxy biochanin A6
-
Substrates: -
Products: -
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?
S-adenosyl-L-methionine + daidzein
S-adenosyl-L-homocysteine + formononetin
-
Substrates: 9% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + deoxyauramycinone
?
-
Substrates: 20% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + epi-frenolicin B
?
-
Substrates: 67% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + fluostatin C 30
?
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + frenolicin B
?
-
Substrates: 78% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + galangin
?
-
Substrates: 7% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + genistein
S-adenosyl-L-homocysteine + 7-methoxygenistein
-
Substrates: 37% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + iodoquinol
?
-
Substrates: 88% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + kaempferol
?
-
Substrates: 39% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + methylcaffeate
?
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + nogalavinone
?
-
Substrates: 76% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + orthosporin
S-adenosyl-L-homocysteine + diaporthin
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + platencin
?
-
Substrates: 26% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + platensimycin
?
-
Substrates: 28% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + purpurin
?
-
Substrates: 13% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + pyramidamycin A
?
-
Substrates: 19% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + pyramidamycin B
?
-
Substrates: 6% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + quercetin
?
-
Substrates: 12% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + quercitrin
?
-
Substrates: 75% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + rabelomycin
?
-
Substrates: 98% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + radicicol
?
-
Substrates: 44% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + rhodomycin D
S-adenosyl-L-homocysteine + 4-O-methoxyrhodomycin D
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + silybin B
?
-
Substrates: 32% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + steffimycin B
?
-
Substrates: 15% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + tiancimycin C
?
-
Substrates: 98% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + tiancimycin F
?
-
Substrates: 98% conversion
Products: -
Products: -
?
S-adenosyl-L-methionine + tiancimycin G
?
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-[(3S)-3-amino-3-(1H-tetrazol-5-yl)propyl](methyl)sulfanium + 2-chloro-4-nitrophenol
S-adenosyl-(3S)-3-amino-3-(1H-tetrazol-5-yl)propane-1-thiol + methyl-2-chloro-4-nitrophenol
Substrates: S-adenosyl-[(3S)-3-amino-3-(1H-tetrazol-5-yl)propyl](methyl)sulfanium is a derivative of AdoMet (S-adenosyl-L-methionine)
Products: -
Products: -
?
S-adenosyl-L-methionine + 13-dihydrocarminomycin
S-adenosyl-L-homocysteine + daunorubicinol
-
Substrates: terminal enzyme in the daunomycin biosynthesis pathway
Products: -
Products: -
?
S-adenosyl-L-methionine + 13-dihydrocarminomycin
S-adenosyl-L-homocysteine + daunorubicinol
-
Substrates: the enzyme shows a slightly higher affinity for carminomycin than for 13-dihydrocarminomycin
Products: -
Products: -
?
S-adenosyl-L-methionine + 13-dihydrocarminomycin
S-adenosyl-L-homocysteine + daunorubicinol
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunomycin
Substrates: with the corresponding aglycones, carminomycinone and 13-dihydrocarminomycinone, as substrates, no methylated products are detected. Other 4-hydroxyanthracyclines such as aklavin and aclacinomycin A, and 4-hydroxyanthracyclinones such as erhodomycinone and aklavinone, are not substrates for the 4-O-methyltransferase. These reaction specificities indicate that glycosylation of the anthracyclinone molecule must occur before 4-O-methylation, which means that 4-O-methylation of carminomycin is probably the terminal step in the biosynthesis of daunomycin, and that daunomycinone is not an intermediate in the pathway
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunomycin
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
-
Substrates: the enzyme shows a slightly higher affinity for carminomycin than for 13-dihydrocarminomycin
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
-
Substrates: -
Products: -
Products: -
?
additional information
?
-
Substrates: the enzyme possesses rather relaxed substrate specificity in regard to modifications in the polyaromatic anthracycline ring system, but it is quite specific with respect to the length of the carbohydrate chain at C-7, accepting only monoglycosides. In addition, DnrK has even been shown to be able to methylate various flavonoids. Discovery of a 10-decarboxylation activity of DnrK
Products: -
Products: -
?
additional information
?
-
Substrates: the enzyme has been reported to methylate various modified anthracyclines and flavonoids as surrogate substrates. 2-Chloro-4-nitrophenol is a substrate of DnrK. High-throughput DnrK colorimetric assay with the surrogate acceptor 2-chloro-4-nitrophenol and the different cosubstrates. No activity with derivative S-(7-deazaadenosyl)-[(3S)-3-amino-3-(1H-tetrazol-5-yl)propyl](methyl)sulfanium
Products: -
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?
additional information
?
-
-
Substrates: 8-methoxyquinoline, orlandin, 4,5,7-trihydroxy-3-phenylcoumarin, 4-hydroxycinnamamide, puerarin, pomiferin, (2S)-naringenin 7-beta-D-glucoside, 4'-hydroxyflavanonequercetin, 5,7,3',4'-tetramethyl ether, tiancimycin A, tetracenomycin C, 6-hydroxy-tetracenomycin C, quinizarin, juglone, andomycinone, landomycin E and himalaquinone G are no substrates
Products: -
Products: -
-
additional information
?
-
-
Substrates: the purified enzyme does not catalyse methylation of the aglycones carminomycinone or 13-dihydrocarminomycinone
Products: -
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?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + 11-deoxy-beta-rhodomycin T
S-adenosyl-L-homocysteine + 4-O-methyl-11-deoxy-beta-rhodomycin T
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + 13-dihydrocarminomycin
S-adenosyl-L-homocysteine + daunorubicinol
-
Substrates: terminal enzyme in the daunomycin biosynthesis pathway
Products: -
Products: -
?
S-adenosyl-L-methionine + 15-demethoxy-aclacinomycin T
S-adenosyl-L-homocysteine + 4-O-methyl-15-decarboxyaclacinomycin T
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunomycin
Substrates: with the corresponding aglycones, carminomycinone and 13-dihydrocarminomycinone, as substrates, no methylated products are detected. Other 4-hydroxyanthracyclines such as aklavin and aclacinomycin A, and 4-hydroxyanthracyclinones such as erhodomycinone and aklavinone, are not substrates for the 4-O-methyltransferase. These reaction specificities indicate that glycosylation of the anthracyclinone molecule must occur before 4-O-methylation, which means that 4-O-methylation of carminomycin is probably the terminal step in the biosynthesis of daunomycin, and that daunomycinone is not an intermediate in the pathway
Products: -
Products: -
?
additional information
?
-
Substrates: the enzyme possesses rather relaxed substrate specificity in regard to modifications in the polyaromatic anthracycline ring system, but it is quite specific with respect to the length of the carbohydrate chain at C-7, accepting only monoglycosides. In addition, DnrK has even been shown to be able to methylate various flavonoids. Discovery of a 10-decarboxylation activity of DnrK
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
-
Substrates: -
Products: -
Products: -
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
-
Substrates: -
Products: -
Products: -
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
design, synthesis, and evaluation of stable, functional AdoMet isosteres that are resistant to the primary contributors to AdoMet degradation (depurination, intramolecular cyclization, and sulfonium epimerization). The AdoMet surrogates to serve as competent enzyme cosubstrates and to bind a prototypical class I model methyltransferase (DnrK) in a manner nearly identical to AdoMet. Half-lives of AdoMet derivatives, overview. Analysis of DnrK ligand-bound structures: DnrK-S-adenosyl-(3S)-3-amino-3-(1H-tetrazol-5-yl)propane-1-thiol and DnrK-S-adenosyl-(3S)-3-amino-3-(1H-tetrazol-5-yl)propane-1-thiol-carminomycin
-
S-adenosyl-L-methionine
S-adenosyl-L-methionine (AdoMet) is an essential enzyme cosubstrate
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
no divalent cation requirement can be demonstrated for the partially purified enzyme
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
homocysteine, adenosine, epsilon-rhodomycinone, daunomycin, and daunomycinone show little or no inhibitory activity
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.335
S-adenosyl-[(3S)-3-amino-3-(1H-tetrazol-5-yl)propyl](methyl)sulfanium
pH 8.0, 37°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.00052
S-adenosyl-[(3S)-3-amino-3-(1H-tetrazol-5-yl)propyl](methyl)sulfanium
pH 8.0, 37°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0015
S-adenosyl-[(3S)-3-amino-3-(1H-tetrazol-5-yl)propyl](methyl)sulfanium
pH 8.0, 37°C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5 - 8
-
pH 7.5: 40% of maximal activity, pH 8.0: optimum. No pH values above pH 8.0 are evaluated due to the inherent instability of S-adenosyl-L-methionine at alkaline pH
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30 - 45
-
30°C: about 90% of maximal activity, 45°C: about 90% of maximal activity
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
Highest Expressing Human Cell Lines
Cell Line LinksPlease wait a moment until the data is sorted. This message will disappear when the data is sorted.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
insertion of a single serine residue to DnrK is sufficient for introduction of a monooxygenation activity. The inserted serine S297 resides in an alpha-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. The shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK
metabolism
physiological function
additional information
the substrate binding site is formed between the C-terminal domain, which has a Rossmann-like alpha/beta-fold typical to nucleotide-binding proteins, and the middle domain
evolution
phylogenetic analysis of the putative SAM-dependent methyltransferases reveals our distinct clades, which are composed of DnrK and RdmB-type proteins and two other groups of sequences. Evolution of anthracycline methyltransferase-like proteins, the phylogenetic analysis suggests that the functional divergence of these proteins has occurred in situ in their respective gene clusters, overview
evolution
the 10-decarboxylation activity of DnrK is the basis of evolution of a RdmB (aclacinomycin 10-hydroxylase RdmB, EC 3.1.1.95, UniProt ID Q54527), an atypical 10-hydroxylase that requires SAM of the rhodomycin pathways, which has 10-hydroxylation ability. Structural comparisons of DnrK and RdmB. phylogenetic tree and substrate specificities, overview
metabolism
the S-adenosyl methionine (SAM)-dependent methyltransferase DnrK is a 4-O-methyltransferase involved in daunorubicin biosynthesis
metabolism
both DnrK and RdmB are postulated to catalyze the decarboxylation of 15-demethoxy-aclacinomycin T as a first step. In DnrK, the open active site allows protonation of the resulting carbanion by solvent molecules and 4-O-methylation to generate 4-O-methyl-15-decarboxyaclacinomycin T. In contrast, RdmB stabilizes the carbanion and catalyzes the formation of an O2-anthracycline caged radical pair in the closed active site. Consequent formation of a peroxyl intermediate and subsequent reduction of the peroxide by intracellular thiols possibly outside the active site of the enzyme yields the 10-hydroxylated product 11-deoxy-beta-rhodomycin T. The reaction sequence leading to the double-product 4-O-methyl-11-deoxy-beta-rhodomycin T is only possible if the RdmB reaction happens first. R1, L-rhodosamine. Formation of the DnrK/RdmB double-reaction product 4-O-methyl-11-deoxy-beta-rhodomycin T must proceed through initial 10-hydroxylation followed by 4-O-methylation
physiological function
the enzyme catalyzes the terminal enzymic step in the formation of daunomycin
physiological function
the enzyme is involved in daunorubicin biosynthesis
physiological function
methyltransferase DnrK is a true methyltransferase that catalyzes the 4-O-methylation of carminomycin in one of the final steps in the biosynthesis of the antitumor drug daunorubicin in Streptomyces peucetius. The enzyme possesses rather relaxed substrate specificity in regard to modifications in the polyaromatic anthracycline ring system, but it is quite specific with respect to the length of the carbohydrate chain at C-7, accepting only monoglycosides. In addition, DnrK has even been shown to be able to methylate various flavonoids
physiological function
the prototypical class I MT DnrK is a model and is the carminomycin 4-O-ethyltransferase that catalyzes a culminating step in the biosynthesis of the anticancer agent daunorubicin in Streptomyces peucetius
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). I2N5E8_STRT9
Streptomyces tsukubensis (strain DSM 42081 / NBRC 108919 / NRRL 18488 / 9993)
365
0
38536
TrEMBL
Secretory Pathway (Reliability: 2)
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in complex with S-adenosyl-L-homocysteine and 4-methylumbelliferone, hanging drop vapor diffusion method, using 1.26 M ammonium sulfate, 0.1 M Tris, pH 8.0, and 0.2 M lithium sulfate
-
purified enzyme mutant DnrK-Ser in complex with aclacinomycin T and S-adenosyl-L-homocysteine, X-ray diffraction structure determination and analysis at 1.9 A resolution
the crystal structure of the ternary complex of this enzyme with the bound products S-adenosyl-L-homocysteine and 4-methoxy-epsilon-rhodomycin T is determined to a 2.35 A resolution, vapor diffusion method at 20°C
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
R302I
site-directed mutagenesis, the mutation greatly reduces the methylation activity on flavonoids
R303K
site-directed mutagenesis, the DnrK R1.2 mutant shows altered substrate specificity, the mutant displays greatly reduced activities for both 4-O-methylation and 10-hydroxylation. The 10-hydroxylation activity is completely lost in the R303Q mutant, whereas only trace activities remain in the R303K mutant when 8 is used as a substrate. The 4-O-methylation activity is also affected, although the mutant still harbors about 10% of its activity
R303Q
site-directed mutagenesis, the DnrK R1.2 mutant shows altered substrate specificity, the mutant displays greatly reduced activities for both 4-O-methylation and 10-hydroxylation. The 10-hydroxylation activity is completely lost in the R303Q mutant. The 4-O-methylation activity is also affected, although the mutant still harbors about 10% of its activity
S297F
site-directed mutagenesis, the DnrK R1.2 mutant shows altered substrate specificity
Y142W
48% of the rate of the recombinant native enzyme with 4-methoxy-epsilon-rhodomycin T
additional information
insertion of a single serine residue at position 297 to DnrK is sufficient for introduction of a monooxygenation activity. The inserted serine S297 resides in an alpha-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. The shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The 10-decarboxylation activity of DnrK is the basis of evolution of a RdmB, an atypical 10-hydroxylase that requires SAM of the rhodomycin pathways, which has 10-hydroxylation ability. For analysis of the origin of the 10-hydroxylation activity in the DnrK R1 chimera, the R1 region is divided into two segments corresponding to the loop region and the following alpha16 helix, which results in two additional mutants denoted as DnrK R1.1 and DnrK R1.2, respectively. The 10-hydroxylation activity can be attributed solely to the alpha16 helix, because the activity of DnrK R1.2 is similar to that of DnrK R1 (i.e. both methylation and hydroxylation of aclacinomycin T and hydroxylation of triglycosylated aclacinomycin A), whereas DnrK R1.1 behaves like native DnrK (i.e. methylation of aclacinomycin T and no activity with triglycosylated aclacinomycin A). Inspection of the amino acid sequences of the R1.2 region reveals that the RdmB sequence contains an additional serine insertion in this area in comparison with DnrK. Fusion of the dimerization domain of DnrK onto the catalytic domain of RdmB generates the enzyme variant RdmB-CT, the activity of RdmB-CT is not altered and the enzyme catalyzes exclusively 10-hydroxylation. Creation of chimeric enzymes by interchanging key subdomain regions ranging from 4-O-methyl-15-decarboxyaclacinomycin T to 4-O-methyl-11-deoxy-beta-rhodomycin T aa to probe the functional differentiation of the enzyme pair
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Streptomyces violaceus pMK100 (epelmycin producer). The transformant produces the hybrid anthracycline antibiotic 7-O-L-rhodosaminyl-4-O-methyl-epsilon-rhodomycinone (4-O-methylepelmycin D) together with host epelmycins when cultured in antibiotic production medium in the presence of thiostrepton. Attempts on production of hybrid 4-O-methylaclarubicin and 4-O-methyl-1-deoxyobelmycin by the transformants of aclarubicin and 1-deoxyobelmycin producers are unsuccessful
-
gene dnrK, recombinant expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
-
production of a new hybrid anthracycline antibiotic. Expression in Streptomyces violaceus pMK100 (epelmycin producer). The transformant produces the hybrid anthracycline antibiotic 7-O-L-hodosaminyl-4-O-methyl-epsilon-rhodomycinone (4-O-methylepelmycin D) together with host epelmycins when cultured in antibiotic production medium in the presence of thiostrepton. Attempts on production of hybrid 4-O-methylaclarubicin and 4-O-methyl-l-deoxyobelmycin by the transformants of aclarubicin and 1-deoxyobelmycin producers are unsuccessful
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Miyamoto, Y.; Ohta, S.; Johdo, O.; Nagamatsu, Y.; Yoshimoto, A.
Production of a new hybrid anthracycline 4-O-methylepelmycin by heterologous expression of dnrK in epelmycin-producing Streptomyces violaceus
J. Antibiot.
53
828-836
2000
Streptomyces peucetius
brenda
Madduri, K.; Torti, F.; Colombo, A.L.; Hutchinson, C.R.
Cloning and sequencing of a gene encoding carminomycin 4-O-methyltransferase from Streptomyces peucetius and its expression in Escherichia coli
J. Bacteriol.
175
3900-3904
1993
Streptomyces peucetius (Q06528), Streptomyces peucetius
brenda
Madduri, K.; Hutchinson, C.R.
Functional characterization and transcriptional analysis of a gene cluster governing early and late steps in daunorubicin biosynthesis in Streptomyces peucetius
J. Bacteriol.
177
3879-3884
1995
Streptomyces peucetius (Q06528)
brenda
Dickens, M.L.; Ye, J.; Strohl, W.R.
Analysis of clustered genes encoding both early and late steps in daunomycin biosynthesis by Streptomyces sp. strain C5
J. Bacteriol.
177
536-543
1995
Streptomyces sp. (Q55216)
brenda
Dickens, M.L.; Priestley, N.D.; Strohl, W.R.
In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to doxorubicin: functions of DauP, DauK, and DoxA
J. Bacteriol.
179
2641-2650
1997
Streptomyces sp.
brenda
Jansson, A.; Koskiniemi, H.; Mntsl, P.; Niemi, J.; Schneider, G.
Crystal structure of a ternary complex of DnrK, a methyltransferase in daunorubicin biosynthesis, with bound products
J. Biol. Chem.
279
41149-41156
2004
Streptomyces peucetius (Q06528), Streptomyces peucetius
brenda
Connors, N.C.; Bartel, P.L.; Strohl, W.R.
Biosynthesis of anthracyclines: carminomycin 4-0-methyltransferase, the terminal enzymic step in the formation of daunomycin
J. Gen. Microbiol.
136
1895-1898
1990
Streptomyces sp. (Q55216)
-
brenda
Connors, N.C.; Strohl, W.R.
Partial purification and properties of carminomycin 4-0-methyltransferase from Streptomyces sp. strain C5
J. Gen. Microbiol.
139
1353-1362
1993
Streptomyces sp.
-
brenda
Huber, T.; Wang, F.; Singh, S.; Johnson, B.; Zhang, J.; Sunkara, M.; Van Lanen, S.; Morris, A.; Phillips, G.; Thorson, J.
Functional AdoMet isosteres resistant to classical AdoMet degradation pathways
ACS Chem. Biol.
11
2484-2491
2016
Streptomyces peucetius (Q06528)
brenda
Grocholski, T.; Yamada, K.; Sinkkonen, J.; Tirkkonen, H.; Niemi, J.; Metsae-Ketelae, M.
Evolutionary trajectories for the functional diversification of anthracycline methyltransferases
ACS Chem. Biol.
14
850-856
2019
Streptomyces peucetius (Q06528)
brenda
Grocholski, T.; Dinis, P.; Niiranen, L.; Niemi, J.; Metsae-Ketelae, M.
Divergent evolution of an atypical S-adenosyl-L-methionine-dependent monooxygenase involved in anthracycline biosynthesis
Proc. Natl. Acad. Sci. USA
112
9866-9871
2015
Streptomyces peucetius (Q06528)
brenda
Jalali, E.; Wang, F.; Overbay, B.R.; Miller, M.D.; Shaaban, K.A.; Ponomareva, L.V.; Ye, Q.; Saghaeiannejad-Esfahani, H.; Bhardwaj, M.; Steele, A.D.; Teijaro, C.N.; Shen, B.; Van Lanen, S.G.; She, Q.B.; Voss, S.R.; Phillips, G.N.; Thorson, J.S.
Biochemical and structural studies of the carminomycin 4-O-methyltransferase DnrK
J. Nat. Prod.
87
798-809
2024
Streptomyces peucetius
brenda
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