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Literature summary extracted from

  • Kuiper, E.G.; Conn, G.L.
    Binding induced RNA conformational changes control substrate recognition and catalysis by the thiostrepton resistance methyltransferase (Tsr) (2014), J. Biol. Chem., 289, 26189-26200.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.230 gene tsr, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)-pLysS Streptomyces azureus

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.230 R162A site-directed mutagenesis, the mutant binds RNA with 100fold weaker affinity than wild-type Tsr Streptomyces azureus
2.1.1.230 R26A site-directed mutagenesis, the mutant is unable to promote the RNase V1-sensitive RNA structural changes, but maintains its interaction with the RNA under certain conditions for the protection of nucleotides G1068 and G1071 from cleavage by RNase T1 Streptomyces azureus

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.1.1.230 additional information stabilizing the RNA tertiary structure, as in RNA mutant U1061A, decreases Tsr binding affinity and catalytic activity independent of RNA structural changes Streptomyces azureus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.1.1.230 Mg2+ required Streptomyces azureus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.1.1.230 S-adenosyl-L-methionine + adenosine1067 in 23S rRNA Streptomyces azureus enzyme Tsr uses the co-substrate AdoMet to methylate the 23 S rRNA, presumably prior to the assembly of the 50 S subunit as the L11 and proposed Tsr binding surfaces are overlapping S-adenosyl-L-homocysteine + 2'-O-methyladenosine1067 in 23S rRNA
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.230 Streptomyces azureus P18644 thiostrepton producer, gene tsr
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Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.230 recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)-pLysS by nickel affinity and heparin affinity chromatography, followed by gel filtration Streptomyces azureus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.230 S-adenosyl-L-methionine + adenosine1067 in 23S rRNA enzyme Tsr uses the co-substrate AdoMet to methylate the 23 S rRNA, presumably prior to the assembly of the 50 S subunit as the L11 and proposed Tsr binding surfaces are overlapping Streptomyces azureus S-adenosyl-L-homocysteine + 2'-O-methyladenosine1067 in 23S rRNA
-
 ?
2.1.1.230 S-adenosyl-L-methionine + adenosine1067 in 23S rRNA 58-nt RNA substrate secondary structure, and key longrange interactions within the 58-nt domain tertiary fold, overview Streptomyces azureus S-adenosyl-L-homocysteine + 2'-O-methyladenosine1067 in 23S rRNA
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 ?

Subunits

EC Number Subunits Comment Organism
2.1.1.230 homodimer each protomer containing an L30e-like amino-terminal domain and a SPOUT methyltransferase family catalytic carboxyl-terminal domain, both enzyme domains are required for high affinity RNA substrate binding, Tsr domain organization, overview Streptomyces azureus

Synonyms

EC Number Synonyms Comment Organism
2.1.1.230 thiostrepton resistance methyltransferase
-
 Streptomyces azureus
2.1.1.230 thiostrepton-resistance methyltransferase
-
 Streptomyces azureus
2.1.1.230 TSR
-
 Streptomyces azureus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.1.1.230 25 37 assay at Streptomyces azureus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.1.1.230 7.5
-
 assay at Streptomyces azureus

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.230 S-adenosyl-L-methionine
-
 Streptomyces azureus

General Information

EC Number General Information Comment Organism
2.1.1.230 malfunction in the absence of the N-terminal domain, Tsr is catalytically inactive despite possessing the intact S-adenosyl-L-methionine binding sites and catalytic center. The Tsr-C-terminal domain dimer binds the RNA 30fold more weakly than the wild-type enzyme and is unable to promote the N-terminal domain-dependent RNA conformational change Streptomyces azureus
2.1.1.230 additional information each protomer of the homodimer containing an L30e-like amino-terminal domain and a SPOUT methyltransferase family catalytic carboxyl-terminal domain, both enzyme domains are required for high affinity RNA substrate binding. The Tsr-C-terminal domain has intrinsic, weak RNA affinity that is necessary to direct the specific high-affinity Tsr-RNA interaction via N-terminal domains, which have no detectable RNA affinity when isolated. The N-terminal domains increase RNA binding affinity and are necessary for catalysis, RNA binding mechanism, overview. The N-terminal domain of Tsr is an essential component of the RNA substrate recognition mechanism by both promoting high affinity RNA binding and activation of catalysis by the C-terminal domain Streptomyces azureus
2.1.1.230 physiological function the thiostrepton producer Streptomyces azureus prevents self-intoxication by expressing the thiostrepton-resistance methyltransferase (Tsr), which methylates the 2'-hydroxyl of 23 S rRNA nucleotide adenosine 1067 within the thiostrepton binding site Streptomyces azureus