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Information on EC 2.1.1.292 - carminomycin 4-O-methyltransferase and Organism(s) Streptomyces peucetius and UniProt Accession Q06528
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2.1.1.292 carminomycin 4-O-methyltransferaseIUBMB 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 .
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Word Map
- 2.1.1.292
- methyltransferases
-
sam-dependent
- s-adenosyl-l-homocysteine
- daunorubicin
- peucetius
-
s-adenosyl-l-methionine-dependent
- synthesis
The taxonomic range for the selected organisms is: Streptomyces peucetius
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
Synonyms
4-o-methyltransferase, class i methyltransferase, carminomycin 4-o-methyltransferase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
PATHWAY SOURCE
PATHWAYS
BRENDA
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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].
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
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
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-
-
?
S-adenosyl-L-methionine + 15-demethoxy-aclacinomycin T
S-adenosyl-L-homocysteine + 4-O-methyl-15-decarboxyaclacinomycin T
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-
-
?
S-adenosyl-L-methionine + 2-chloro-4-nitrophenol
S-adenosyl-L-homocysteine + methyl-2-chloro-4-nitrophenol
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-
-
?
S-adenosyl-L-methionine + aclacinomycin T
S-adenosyl-L-homocysteine + triglycosylated aclacinomycin A
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-
-
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
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-
-
?
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
S-adenosyl-[(3S)-3-amino-3-(1H-tetrazol-5-yl)propyl](methyl)sulfanium is a derivative of AdoMet (S-adenosyl-L-methionine)
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-
?
additional information
?
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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
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-
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additional information
?
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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
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
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
-
-
-
?
S-adenosyl-L-methionine + 15-demethoxy-aclacinomycin T
S-adenosyl-L-homocysteine + 4-O-methyl-15-decarboxyaclacinomycin T
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-
-
?
S-adenosyl-L-methionine + carminomycin
S-adenosyl-L-homocysteine + daunorubicin
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-
-
?
additional information
?
-
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
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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
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S-adenosyl-L-methionine
S-adenosyl-L-methionine (AdoMet) is an essential enzyme cosubstrate
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
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
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
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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
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
metabolism
the S-adenosyl methionine (SAM)-dependent methyltransferase DnrK is a 4-O-methyltransferase involved in daunorubicin biosynthesis
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
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). PDB
SCOP
CATH
UNIPROT
ORGANISM
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene dnrK, recombinant expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)
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
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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
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
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
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)
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
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)
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)
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