Cloned (Comment) | Organism |
---|---|
gene tsr, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)-pLysS | Streptomyces azureus |
Protein Variants | Comment | Organism |
---|---|---|
R162A | site-directed mutagenesis, the mutant binds RNA with 100fold weaker affinity than wild-type Tsr | Streptomyces azureus |
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 | Comment | Organism | Structure |
---|---|---|---|
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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Streptomyces azureus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | UniProt | Comment | Textmining |
---|---|---|---|
Streptomyces azureus | P18644 | thiostrepton producer, gene tsr | - |
Purification (Comment) | Organism |
---|---|
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | - |
? | |
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 | - |
? |
Subunits | Comment | Organism |
---|---|---|
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 | Comment | Organism |
---|---|---|
thiostrepton resistance methyltransferase | - |
Streptomyces azureus |
thiostrepton-resistance methyltransferase | - |
Streptomyces azureus |
TSR | - |
Streptomyces azureus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | 37 | assay at | Streptomyces azureus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Streptomyces azureus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
S-adenosyl-L-methionine | - |
Streptomyces azureus |
General Information | Comment | Organism |
---|---|---|
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 |
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 |
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 |