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Literature summary for 2.1.1.221 extracted from
- Insights into catalytic and tRNA recognition mechanism of the dual-specific tRNA methyltransferase from Thermococcus kodakarensis (2019), Genes (Basel), 10, 100 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene TK0422, sequence comparisons, recombinant expression of N-terminally His6-tagged protein in Escherichia coli | Thermococcus kodakarensis |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D104A | site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type | Thermococcus kodakarensis |
| D104A/E115Q/D245A | site-directed mutagenesis, almost inactive mutant | Thermococcus kodakarensis |
| D104N | site-directed mutagenesis, the mutant shows slightly increased activity compared to wild-type | Thermococcus kodakarensis |
| D104N/D206N/D245N | site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type | Thermococcus kodakarensis |
| D104N/D206N/D245N/E115Q | site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type | Thermococcus kodakarensis |
| D206A | site-directed mutagenesis, the enzyme activity is modestly reduced compared to wild-type | Thermococcus kodakarensis |
| D206A/D245A | site-directed mutagenesis, the enzyme activity is abolished in the double mutant | Thermococcus kodakarensis |
| D206N | site-directed mutagenesis, the mutant shows moderately reduced activity compared to wild-type | Thermococcus kodakarensis |
| D245A | site-directed mutagenesis, the enzyme activity is modestly reduced compared to wild-type | Thermococcus kodakarensis |
| D245N | site-directed mutagenesis, the mutation has no significant effect on the A-preference for TktRNAAsp, but exhibits a modest, but shows about 4fold reduced G-preference activity compared to wild-type | Thermococcus kodakarensis |
| E115Q | site-directed mutagenesis, the mutant shows moderately reduced activity compared to wild-type | Thermococcus kodakarensis |
| G202R | site-directed mutagenesis, the mutant is nearly inactive, nearly complete loss of both m1G9 and m1A9 activity | Thermococcus kodakarensis |
| G242R | site-directed mutagenesis, the mutant shows unaltered activity | Thermococcus kodakarensis |
| Q122A | site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type | Thermococcus kodakarensis |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | enzyme kinetic analysis and modeling | Thermococcus kodakarensis |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required, the two tRNAs from Saccharomyces cerevisiae requires a much higher Mg2+ concentration (6-10 mM in the assay) for maximal TkTrm10 activity compared to the two Thermococcus kodakarensis tRNAs, for which maximal activity is observed at about 1 mM Mg2+ or less. Similar trends are exhibited for both m1G9 and m1A9 reactions, indicating that the identity of the target purine does not affect the observed metal dependencies | Thermococcus kodakarensis |  |
| additional information | no activity is detected in the absence of metal | Thermococcus kodakarensis |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| S-adenosyl-L-methionine + guanine9 in tRNA | Thermococcus kodakarensis | - |
S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNA | - |
? | |
| S-adenosyl-L-methionine + guanine9 in tRNA | Thermococcus kodakarensis ATCC BAA-918 | - |
S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNA | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Thermococcus kodakarensis | Q5JD38 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant N-terminally His6-tagged protein from Escherichia coli by nickel affinity chromatography and dialysis | Thermococcus kodakarensis |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | usage of [alpha-32P]-labeled tRNA substrates. The enzyme shows activity with both guanine9 and adenine9 containing tRNAs for methylation on N1. Bifunctional enzymes (catalyzing both m1A9 and m1G9) share the same rate-determining step for methylation as the monofunctional enzyme, these enzymes would also exhibit a different pattern of pH dependence for the two methylation reactions because of the difference in N1 pKa between adenine versus guanine | Thermococcus kodakarensis | ? | - |
- |
|
| additional information | usage of [alpha-32P]-labeled tRNA substrates. The enzyme shows activity with both guanine9 and adenine9 containing tRNAs for methylation on N1. Bifunctional enzymes (catalyzing both m1A9 and m1G9) share the same rate-determining step for methylation as the monofunctional enzyme, these enzymes would also exhibit a different pattern of pH dependence for the two methylation reactions because of the difference in N1 pKa between adenine versus guanine | Thermococcus kodakarensis ATCC BAA-918 | ? | - |
- |
|
| S-adenosyl-L-methionine + guanine9 in tRNA | - |
Thermococcus kodakarensis | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNA | - |
? | |
| S-adenosyl-L-methionine + guanine9 in tRNA | - |
Thermococcus kodakarensis ATCC BAA-918 | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNA | - |
? | |
| S-adenosyl-L-methionine + guanine9 in tRNAArg | tRNA substrate from Thermococcus kodakarensis | Thermococcus kodakarensis | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAArg | - |
? | |
| S-adenosyl-L-methionine + guanine9 in tRNAArg | tRNA substrate from Thermococcus kodakarensis | Thermococcus kodakarensis ATCC BAA-918 | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAArg | - |
? | |
| S-adenosyl-L-methionine + guanine9 in tRNAGly | tRNA substrate from Saccharomyces cerevisiae | Thermococcus kodakarensis | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAGly | - |
? | |
| S-adenosyl-L-methionine + guanine9 in tRNAGly | tRNA substrate from Saccharomyces cerevisiae | Thermococcus kodakarensis ATCC BAA-918 | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAGly | - |
? | |
| S-adenosyl-L-methionine + guanine9 in tRNAPhe | tRNA substrate from Saccharomyces cerevisiae | Thermococcus kodakarensis | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAPhe | - |
? | |
| S-adenosyl-L-methionine + guanine9 in tRNAPhe | tRNA substrate from Saccharomyces cerevisiae | Thermococcus kodakarensis ATCC BAA-918 | S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAPhe | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| ? | x * 44000, recombinant His6-atgged enzyme, SDS-PAGE | Thermococcus kodakarensis |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| m1R9-specific TkTrm10 | - |
Thermococcus kodakarensis |
| More | see also EC 2.1.1.218 | Thermococcus kodakarensis |
| TkTrm10 | - |
Thermococcus kodakarensis |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 40 | 50 | assay at, with Saccharomyces cervisiae tRNA and Thermococcus kodakarensis tRNA, respectively | Thermococcus kodakarensis |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0002 | - |
guanine9 in tRNAArg | pH 8.0, 50°C, recombinant enzyme, tRNA substrate from Thermococcus kodakarensis | Thermococcus kodakarensis | |
| 0.0025 | - |
guanine9 in tRNAGly | pH 8.0, 40°C, recombinant enzyme, tRNA substrate from Saccharomyces cerevisiae | Thermococcus kodakarensis | |
| 0.0232 | - |
guanine9 in tRNAPhe | pH 8.0, 40°C, recombinant enzyme, tRNA substrate from Saccharomyces cerevisiae | Thermococcus kodakarensis |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Thermococcus kodakarensis |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| additional information | - |
pH-rate profiles for the two activities catalyzed by the bifunctional methyltransferase TkTrm10, cf. EC 2.1.1.218 | Thermococcus kodakarensis |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| S-adenosyl-L-methionine | - |
Thermococcus kodakarensis | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | residue G202 is important for catalytic activity regardless of the target purine of the tRNA species to be modified | Thermococcus kodakarensis |
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Release 2023.1 (January 2023)
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