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Literature summary extracted from

  • Krishnamohan, A.; Dodbele, S.; Jackman, J.
    Insights into catalytic and tRNA recognition mechanism of the dual-specific tRNA methyltransferase from Thermococcus kodakarensis (2019), Genes (Basel), 10, 100 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.218 gene TK0422, sequence comparisons, recombinant expression of N-terminally His6-tagged protein in Escherichia coli Thermococcus kodakarensis
2.1.1.221 gene TK0422, sequence comparisons, recombinant expression of N-terminally His6-tagged protein in Escherichia coli Thermococcus kodakarensis

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.218 D104A site-directed mutagenesis, the mutant shows moderately reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.218 D104A/E115Q/D245A site-directed mutagenesis, almost inactive mutant Thermococcus kodakarensis
2.1.1.218 D104N site-directed mutagenesis, the mutant shows slightly reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.218 D104N/D206N/D245N site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.218 D104N/D206N/D245N/E115Q site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.218 D206A site-directed mutagenesis, the enzyme activity is modestly reduced compared to wild-type Thermococcus kodakarensis
2.1.1.218 D206A/D245A site-directed mutagenesis, the enzyme activity is abolished in the double mutant Thermococcus kodakarensis
2.1.1.218 D206N site-directed mutagenesis, the mutant shows moderately reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.218 D245A site-directed mutagenesis, the enzyme activity is modestly reduced compared to wild-type Thermococcus kodakarensis
2.1.1.218 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
2.1.1.218 E115Q site-directed mutagenesis, the mutant shows moderately reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.218 G202R site-directed mutagenesis, the mutant is nearly inactive, nearly complete loss of both m1G9 and m1A9 activity Thermococcus kodakarensis
2.1.1.218 G242R site-directed mutagenesis, the mutant shows unaltered activity Thermococcus kodakarensis
2.1.1.218 Q122A site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.221 D104A site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.221 D104A/E115Q/D245A site-directed mutagenesis, almost inactive mutant Thermococcus kodakarensis
2.1.1.221 D104N site-directed mutagenesis, the mutant shows slightly increased activity compared to wild-type Thermococcus kodakarensis
2.1.1.221 D104N/D206N/D245N site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.221 D104N/D206N/D245N/E115Q site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.221 D206A site-directed mutagenesis, the enzyme activity is modestly reduced compared to wild-type Thermococcus kodakarensis
2.1.1.221 D206A/D245A site-directed mutagenesis, the enzyme activity is abolished in the double mutant Thermococcus kodakarensis
2.1.1.221 D206N site-directed mutagenesis, the mutant shows moderately reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.221 D245A site-directed mutagenesis, the enzyme activity is modestly reduced compared to wild-type Thermococcus kodakarensis
2.1.1.221 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
2.1.1.221 E115Q site-directed mutagenesis, the mutant shows moderately reduced activity compared to wild-type Thermococcus kodakarensis
2.1.1.221 G202R site-directed mutagenesis, the mutant is nearly inactive, nearly complete loss of both m1G9 and m1A9 activity Thermococcus kodakarensis
2.1.1.221 G242R site-directed mutagenesis, the mutant shows unaltered activity Thermococcus kodakarensis
2.1.1.221 Q122A site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type Thermococcus kodakarensis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.1.1.218 additional information
-
 additional information enzyme kinetic analysis and modeling Thermococcus kodakarensis
2.1.1.221 additional information
-
 additional information enzyme kinetic analysis and modeling Thermococcus kodakarensis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.1.1.218 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
2.1.1.218 additional information no activity is detected in the absence of metal Thermococcus kodakarensis
2.1.1.221 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
2.1.1.221 additional information no activity is detected in the absence of metal Thermococcus kodakarensis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.1.1.218 S-adenosyl-L-methionine + adenine9 in tRNA Thermococcus kodakarensis
-
 S-adenosyl-L-homocysteine + N1-methyladenine9 in tRNA
-
 ?
2.1.1.221 S-adenosyl-L-methionine + guanine9 in tRNA Thermococcus kodakarensis
-
 S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNA
-
 ?
2.1.1.221 S-adenosyl-L-methionine + guanine9 in tRNA Thermococcus kodakarensis ATCC BAA-918
-
 S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNA
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.218 Thermococcus kodakarensis Q5JD38
-
-
2.1.1.218 Thermococcus kodakarensis ATCC BAA-918 Q5JD38
-
-
2.1.1.221 Thermococcus kodakarensis Q5JD38
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.218 recombinant N-terminally His6-tagged protein from Escherichia coli by nickel affinity chromatography and dialysis Thermococcus kodakarensis
2.1.1.221 recombinant N-terminally His6-tagged protein from Escherichia coli by nickel affinity chromatography and dialysis Thermococcus kodakarensis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.218 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 ?
-
-
2.1.1.218 S-adenosyl-L-methionine + adenine9 in tRNA
-
 Thermococcus kodakarensis S-adenosyl-L-homocysteine + N1-methyladenine9 in tRNA
-
 ?
2.1.1.218 S-adenosyl-L-methionine + adenine9 in tRNAPhe tRNA substrate from Saccharomyces cerevisiae Thermococcus kodakarensis S-adenosyl-L-homocysteine + N1-methyladenine9 in tRNAPhe
-
 ?
2.1.1.218 S-adenosyl-L-methionine + adenine9 in tRNAThr tRNA substrate from Thermococcus kodakarensis Thermococcus kodakarensis S-adenosyl-L-homocysteine + N1-methyladenine9 in tRNAThr
-
 ?
2.1.1.221 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 ?
-
-
2.1.1.221 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 ?
-
-
2.1.1.221 S-adenosyl-L-methionine + guanine9 in tRNA
-
 Thermococcus kodakarensis S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNA
-
 ?
2.1.1.221 S-adenosyl-L-methionine + guanine9 in tRNA
-
 Thermococcus kodakarensis ATCC BAA-918 S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNA
-
 ?
2.1.1.221 S-adenosyl-L-methionine + guanine9 in tRNAArg tRNA substrate from Thermococcus kodakarensis Thermococcus kodakarensis S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAArg
-
 ?
2.1.1.221 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
-
 ?
2.1.1.221 S-adenosyl-L-methionine + guanine9 in tRNAGly tRNA substrate from Saccharomyces cerevisiae Thermococcus kodakarensis S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAGly
-
 ?
2.1.1.221 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
-
 ?
2.1.1.221 S-adenosyl-L-methionine + guanine9 in tRNAPhe tRNA substrate from Saccharomyces cerevisiae Thermococcus kodakarensis S-adenosyl-L-homocysteine + N1-methylguanine9 in tRNAPhe
-
 ?
2.1.1.221 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

EC Number Subunits Comment Organism
2.1.1.218 ? x * 44000, recombinant His6-tagged enzyme, SDS-PAGE Thermococcus kodakarensis
2.1.1.221 ? x * 44000, recombinant His6-atgged enzyme, SDS-PAGE Thermococcus kodakarensis

Synonyms

EC Number Synonyms Comment Organism
2.1.1.218 m1R9-specific TkTrm10
-
 Thermococcus kodakarensis
2.1.1.218 More see also EC 2.1.1.221 Thermococcus kodakarensis
2.1.1.218 TkTrm10
-
 Thermococcus kodakarensis
2.1.1.221 m1R9-specific TkTrm10
-
 Thermococcus kodakarensis
2.1.1.221 More see also EC 2.1.1.218 Thermococcus kodakarensis
2.1.1.221 TkTrm10
-
 Thermococcus kodakarensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.1.1.218 40 50 assay at, with Saccharomyces cervisiae tRNA and Thermococcus kodakarensis tRNA, respectively Thermococcus kodakarensis
2.1.1.221 40 50 assay at, with Saccharomyces cervisiae tRNA and Thermococcus kodakarensis tRNA, respectively Thermococcus kodakarensis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.1.1.218 0.002
-
 adenine9 in tRNAThr pH 8.0, 50°C, recombinant enzyme, tRNA substrate from Thermococcus kodakarensis Thermococcus kodakarensis
2.1.1.218 0.0093
-
 adenine9 in tRNAPhe pH 8.0, 40°C, recombinant enzyme, tRNA substrate from Saccharomyces cerevisiae Thermococcus kodakarensis
2.1.1.221 0.0002
-
 guanine9 in tRNAArg pH 8.0, 50°C, recombinant enzyme, tRNA substrate from Thermococcus kodakarensis Thermococcus kodakarensis
2.1.1.221 0.0025
-
 guanine9 in tRNAGly pH 8.0, 40°C, recombinant enzyme, tRNA substrate from Saccharomyces cerevisiae Thermococcus kodakarensis
2.1.1.221 0.0232
-
 guanine9 in tRNAPhe pH 8.0, 40°C, recombinant enzyme, tRNA substrate from Saccharomyces cerevisiae Thermococcus kodakarensis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.1.1.218 8
-
 assay at Thermococcus kodakarensis
2.1.1.221 8
-
 assay at Thermococcus kodakarensis

pH Range

EC Number pH Minimum pH Maximum Comment Organism
2.1.1.218 additional information
-
 pH-rate profiles for the two activities catalyzed by the bifunctional methyltransferase TkTrm10, cf. EC 2.1.1.221 Thermococcus kodakarensis
2.1.1.221 additional information
-
 pH-rate profiles for the two activities catalyzed by the bifunctional methyltransferase TkTrm10, cf. EC 2.1.1.218 Thermococcus kodakarensis

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.218 S-adenosyl-L-methionine
-
 Thermococcus kodakarensis
2.1.1.221 S-adenosyl-L-methionine
-
 Thermococcus kodakarensis

General Information

EC Number General Information Comment Organism
2.1.1.218 additional information residue G202 is important for catalytic activity regardless of the target purine of the tRNA species to be modified Thermococcus kodakarensis
2.1.1.221 additional information residue G202 is important for catalytic activity regardless of the target purine of the tRNA species to be modified Thermococcus kodakarensis