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Literature summary extracted from

  • Wang, C.; Jia, Q.; Zeng, J.; Chen, R.; Xie, W.
    Structural insight into the methyltransfer mechanism of the bifunctional Trm5 (2017), Sci. Adv., 3, e1700195 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.228 gene trm5a, recombinant expression of His6-tagged wild-type and mutant enzymes Pyrococcus abyssi
2.1.1.282 gene trm5a, recombinant expression of wild-type and mutant enzymes Pyrococcus abyssi

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.1.1.228 analysis of enzyme complex structures of Trm5b (MjTrm5b, PDB IDs 2YX1 and 3AY0) from Methanococcus jannaschii Methanocaldococcus jannaschii
2.1.1.228 purified recombinant apo-PaTrm5a and PaTrm5a in complex with tRNAPhe, with SAM, and as PaTrm5a-tRNAPhe (imG-14)-SAH ternary complex, sitting drop vapor diffusion method, 6 mg/ml recombinant His6-tagged enzyme protein is mixed with tRNAPhe at a molar ratio of 1:0.4 in a buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, and 1.5 mM SAH or SAM, and equilibration against a reservoir solution containing 45% 2-methyl-2,4-pentanediol, 100 mM MES, pH 6.5, and 200 mM NH4OAc, 1 week, X-ray diffraction structure determination and analysis at 2.6-3.2 A resolution, molecular replacement using complex structure of the MjTrm5b-tRNACys-SAM complex (PDB ID 2ZZN) as the search model, modelling Pyrococcus abyssi
2.1.1.282 purified recombinant apo-PaTrm5a and PaTrm5a in complex with tRNAPhe, and PaTrm5a-tRNAPhe (imG-14)-SAH ternary complex, X-ray diffraction structure determination and analysis Pyrococcus abyssi

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.228 D243A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.228 E213A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.228 H128A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.228 R133A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.228 R135A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.228 R174A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.228 Y318A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.282 D243A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.282 E213A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.282 H128A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.282 R133A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.282 R135A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.282 R174A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi
2.1.1.282 Y318A site-directed mutagenesis, substrate binding compared to wild-type enzyme Pyrococcus abyssi

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.1.1.228 Mg2+ required Methanocaldococcus jannaschii
2.1.1.228 Mg2+ required Pyrococcus abyssi
2.1.1.282 Mg2+ required Pyrococcus abyssi

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.1.1.228 additional information Pyrococcus abyssi bifunctional Trm5a from Pyrococcus abyssi (PaTrm5a) catalyses not only the methylation of N1, but also the further methylation of C7 on 4 demethylwyosine at position 37 to produce isowyosine (EC 2.1.1.228 and EC 2.1.1.282, respectively) ?
-
-
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAPhe Methanocaldococcus jannaschii
-
 S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAPhe Pyrococcus abyssi
-
 S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAPhe Methanocaldococcus jannaschii DSM 2661
-
 S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe
-
 ?
2.1.1.282 additional information Pyrococcus abyssi bifunctional Trm5a from Pyrococcus abyssi (PaTrm5a) catalyses not only the methylation of N1, but also the further methylation of C7 on 4 demethylwyosine at position 37 to produce isowyosine (EC 2.1.1.228 and EC 2.1.1.282, respectively) ?
-
-
2.1.1.282 S-adenosyl-L-methionine + 7-[(3S)-(3-amino-3-carboxypropyl)]-4-demethylwyosine37 in tRNAPhe Pyrococcus abyssi
-
 S-adenosyl-L-homocysteine + 7-[(3S)-(3-amino-3-carboxypropyl)]wyosine37 in tRNAPhe
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.228 Methanocaldococcus jannaschii Q58293
-
-
2.1.1.228 Methanocaldococcus jannaschii DSM 2661 Q58293
-
-
2.1.1.228 Pyrococcus abyssi Q9V2G1
-
-
2.1.1.282 Pyrococcus abyssi Q9V2G1
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.228 recombinant His6-tagged wild-type and mutant enzymes Pyrococcus abyssi
2.1.1.282 recombinant wild-type and mutant enzymes Pyrococcus abyssi

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.228 additional information bifunctional Trm5a from Pyrococcus abyssi (PaTrm5a) catalyses not only the methylation of N1, but also the further methylation of C7 on 4 demethylwyosine at position 37 to produce isowyosine (EC 2.1.1.228 and EC 2.1.1.282, respectively) Pyrococcus abyssi ?
-
-
2.1.1.228 additional information structural basis for substrate recognition, the D1 domain of the enzyme undergoes large conformational changes upon the binding of tRNA, the enzyme recognizes the overall shape of tRNA, overview. Enzyme-substrate interactions in the catalytic domain, D1 domain ofMjTrm5b transitions, overview Methanocaldococcus jannaschii ?
-
-
2.1.1.228 additional information structural basis for substrate recognition, the D1 domain of the enzyme undergoes large conformational changes upon the binding of tRNA. The enzyme recognizes the overall shape of tRNA. PaTrm5a adopts distinct open conformations before and after the binding of tRNA. Enzyme-substrate interactions in the catalytic domain. The anticodon interactions mostly concentrate on the A36-G37-A38 triplet. Proposed reaction mechanism of Trm5a with modified yeast tRNAPhe, overview Pyrococcus abyssi ?
-
-
2.1.1.228 additional information structural basis for substrate recognition, the D1 domain of the enzyme undergoes large conformational changes upon the binding of tRNA, the enzyme recognizes the overall shape of tRNA, overview. Enzyme-substrate interactions in the catalytic domain, D1 domain ofMjTrm5b transitions, overview Methanocaldococcus jannaschii DSM 2661 ?
-
-
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAPhe
-
 Methanocaldococcus jannaschii S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAPhe
-
 Pyrococcus abyssi S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe
-
 ?
2.1.1.228 S-adenosyl-L-methionine + guanine37 in tRNAPhe
-
 Methanocaldococcus jannaschii DSM 2661 S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe
-
 ?
2.1.1.282 additional information bifunctional Trm5a from Pyrococcus abyssi (PaTrm5a) catalyses not only the methylation of N1, but also the further methylation of C7 on 4 demethylwyosine at position 37 to produce isowyosine (EC 2.1.1.228 and EC 2.1.1.282, respectively) Pyrococcus abyssi ?
-
-
2.1.1.282 additional information structural basis for substrate recognition, the D1 domain of the enzyme undergoes large conformational changes upon the binding of tRNA., the enzyme recognizes the overall shape of tRNA. PaTrm5a adopts distinct open conformations before and after the binding of tRNA. Enzyme-substrate interactions in the catalytic domain. The anticodon interactions mostly concentrate on the A36-G37-A38 triplet. Proposed reaction mechanism of Trm5a with modified yeast tRNAPhe, overview Pyrococcus abyssi ?
-
-
2.1.1.282 S-adenosyl-L-methionine + 7-[(3S)-(3-amino-3-carboxypropyl)]-4-demethylwyosine37 in tRNAPhe
-
 Pyrococcus abyssi S-adenosyl-L-homocysteine + 7-[(3S)-(3-amino-3-carboxypropyl)]wyosine37 in tRNAPhe
-
 ?
2.1.1.282 S-adenosyl-L-methionine + 7-[(3S)-(3-amino-3-carboxypropyl)]-4-demethylwyosine37 in tRNAPhe imG-14 Pyrococcus abyssi S-adenosyl-L-homocysteine + 7-[(3S)-(3-amino-3-carboxypropyl)]wyosine37 in tRNAPhe
-
 ?

Subunits

EC Number Subunits Comment Organism
2.1.1.228 More substrate interactions with the catalytic region in the PaTrm5a-tRNAPhe-SAH ternary complex, overview Pyrococcus abyssi
2.1.1.282 More substrate interactions with the catalytic region in the PaTrm5a-tRNAPhe-(imG-14)-SAH ternary complex, overview. In the model, imG-14 is flipped into a hydrophobic pocket formed by Phe165, Phe284, Tyr318, Met170, Tyr197, and the 260PTPK263 fragment Pyrococcus abyssi

Synonyms

EC Number Synonyms Comment Organism
2.1.1.228 MjTrm5b
-
 Methanocaldococcus jannaschii
2.1.1.228 More see also EC 2.1.1.282 Pyrococcus abyssi
2.1.1.228 PaTrm5a
-
 Pyrococcus abyssi
2.1.1.228 TAW22
-
 Pyrococcus abyssi
2.1.1.228 Trm5a
-
 Pyrococcus abyssi
2.1.1.228 trm5b
-
 Methanocaldococcus jannaschii
2.1.1.282 More see also EC 2.1.1.228 Pyrococcus abyssi
2.1.1.282 PaTrm5a
-
 Pyrococcus abyssi
2.1.1.282 TAW22
-
 Pyrococcus abyssi
2.1.1.282 Trm5a
-
 Pyrococcus abyssi

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.1.1.228 37
-
 assay at Methanocaldococcus jannaschii
2.1.1.228 37
-
 assay at Pyrococcus abyssi
2.1.1.282 37
-
 assay at Pyrococcus abyssi

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.1.1.228 8
-
 assay at Methanocaldococcus jannaschii
2.1.1.228 8
-
 assay at Pyrococcus abyssi
2.1.1.282 8
-
 assay at Pyrococcus abyssi

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.228 S-adenosyl-L-methionine
-
 Methanocaldococcus jannaschii
2.1.1.228 S-adenosyl-L-methionine
-
 Pyrococcus abyssi
2.1.1.282 S-adenosyl-L-methionine
-
 Pyrococcus abyssi

General Information

EC Number General Information Comment Organism
2.1.1.228 malfunction deletion of the D1 domain greatly reduces the affinity and activity of PaTrm5a toward its RNA substrate Pyrococcus abyssi
2.1.1.228 additional information structure comparison of the Pyrococcus abyssii Trm5a enzyme structure (PDB IDs 5HJJ and 5WT1) with the structure of its orthologue Trm5b (MjTrm5b, PDB IDs 2YX1 and 3AY0) from Methanococcus jannaschii, overview Methanocaldococcus jannaschii
2.1.1.228 additional information structure comparison of the Pyrococcus abyssii Trm5a enzyme structure (PDB IDs 5HJJ and 5WT1) with the structure of its orthologue Trm5b (MjTrm5b, PDB IDs 2YX1 and 3AY0) from Methanococcus jannaschii, overview Pyrococcus abyssi
2.1.1.228 physiological function the methyltransferase Trm5a from Pyrococcus abyssi (PaTrm5a) plays a key role in this hypermodification process in generating m1G37 (EC 2.1.1.228) and imG2 (EC 2.1.1.282), two products of the wyosine biosynthetic pathway, through two methyl transfers to distinct substrates Pyrococcus abyssi
2.1.1.282 malfunction deletion of the D1 domain greatly reduces the affinity and activity of PaTrm5a toward its RNA substrate Pyrococcus abyssi
2.1.1.282 additional information structure comparison of the Pyrococcus abyssii Trm5a enzyme structure (PDB ID 5WT1) with the structure of its orthologue Trm5b (MjTrm5b, PDB IDs 2YX1 and 3AY0) from Methanococcus jannaschii, overview Pyrococcus abyssi
2.1.1.282 physiological function the methyltransferase Trm5a from Pyrococcus abyssi (PaTrm5a) plays a key role in this hypermodification process in generating m1G37 (EC 2.1.1.228) and imG2 (EC 2.1.1.282), two products of the wyosine biosynthetic pathway, through two methyl transfers to distinct substrates Pyrococcus abyssi