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Literature summary for 2.1.1.292 extracted from
- Divergent evolution of an atypical S-adenosyl-L-methionine-dependent monooxygenase involved in anthracycline biosynthesis (2015), Proc. Natl. Acad. Sci. USA, 112, 9866-9871 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| phylogenetic tree | Streptomyces peucetius |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| 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 | Streptomyces peucetius |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| 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 | Streptomyces peucetius |
| R302I | site-directed mutagenesis, the mutation greatly reduces the methylation activity on flavonoids | Streptomyces peucetius |
| 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 | Streptomyces peucetius |
| 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 | Streptomyces peucetius |
| S297F | site-directed mutagenesis, the DnrK R1.2 mutant shows altered substrate specificity | Streptomyces peucetius |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | 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. Discovery of a 10-decarboxylation activity of DnrK | ? | - |
- |
 |
| S-adenosyl-L-methionine + 11-deoxy-beta-rhodomycin T | Streptomyces peucetius | - |
S-adenosyl-L-homocysteine + 4-O-methyl-11-deoxy-beta-rhodomycin T | - |
? | |
| S-adenosyl-L-methionine + 15-demethoxy-aclacinomycin T | Streptomyces peucetius | - |
S-adenosyl-L-homocysteine + 4-O-methyl-15-decarboxyaclacinomycin T | - |
? | |
| S-adenosyl-L-methionine + carminomycin | Streptomyces peucetius | - |
S-adenosyl-L-homocysteine + daunorubicin | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Streptomyces peucetius | Q06528 | - |
- |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 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 | Streptomyces peucetius | ? | - |
- |
|
| S-adenosyl-L-methionine + 11-deoxy-beta-rhodomycin T | - |
Streptomyces peucetius | S-adenosyl-L-homocysteine + 4-O-methyl-11-deoxy-beta-rhodomycin T | - |
? | |
| S-adenosyl-L-methionine + 15-demethoxy-aclacinomycin T | - |
Streptomyces peucetius | S-adenosyl-L-homocysteine + 4-O-methyl-15-decarboxyaclacinomycin T | - |
? | |
| S-adenosyl-L-methionine + aclacinomycin T | - |
Streptomyces peucetius | S-adenosyl-L-homocysteine + triglycosylated aclacinomycin A | - |
? | |
| S-adenosyl-L-methionine + carminomycin | - |
Streptomyces peucetius | S-adenosyl-L-homocysteine + daunorubicin | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| 4-O-methyltransferase | - |
Streptomyces peucetius |
| anthracycline methyltransferase | - |
Streptomyces peucetius |
| DnrK | - |
Streptomyces peucetius |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
assay at | Streptomyces peucetius |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| S-adenosyl-L-methionine | - |
Streptomyces peucetius | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| 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 | Streptomyces peucetius |
| 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 | Streptomyces peucetius |
| 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 | Streptomyces peucetius |
| 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 | Streptomyces peucetius |
| 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 | Streptomyces peucetius |
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