Literature summary extracted from

Wang, C.; Jia, Q.; Chen, R.; Wei, Y.; Li, J.; Ma, J.; Xie, W.
Crystal structures of the bifunctional tRNA methyltransferase Trm5a (2016), Sci. Rep., 6, 33553 .

Cloned(Commentary)

EC Number	Cloned (Comment)	Organism
2.1.1.228	gene trm5a, sequence comparisons, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Pyrococcus abyssi
2.1.1.282	gene trm5a, sequence comparisons, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Pyrococcus abyssi

Crystallization (Commentary)

EC Number	Crystallization (Comment)	Organism
2.1.1.228	purified recombinant PaTrm5a in apo form and in complex with various SAM analogues, mixing PaTrm5a with 1.5 mM SAH or SAM at a protein: ligand molar ratio of 1:3, sitting drop vapor diffusion method, mixing 20 mg/ml protein solution in a 1:1 ratio with well solution containing w/v PEG 3350, 100 mM HEPES, pH 7.5, 100 mM Ca(OAc)2 and 100 mM KCl, method optimmization, 25°C, X-ray diffraction structure determination and analysis at 1.76-2.20 A resolution	Pyrococcus abyssi
2.1.1.282	purified recombinant PaTrm5a in apo form and in complex with various SAM analogues, mixing PaTrm5a with 1.5 mM SAH or SAM at a protein: ligand molar ratio of 1:3, sitting drop vapor diffusion method, mixing 20 mg/ml protein solution in a 1:1 ratio with well solution containing w/v PEG 3350, 100 mM HEPES, pH 7.5, 100 mM Ca(OAc)2 and 100 mM KCl., method optimmization, 25°C, X-ray diffraction structure determination and analysis at 1.76-2.20 A resolution	Pyrococcus abyssi

Protein Variants

EC Number	Protein Variants	Comment	Organism
2.1.1.228	C301S/C308S/C326S	site-directed mutagenesis	Pyrococcus abyssi
2.1.1.228	V21C/C301S/C308S/K314C/C326S	site-directed mutagenesis	Pyrococcus abyssi
2.1.1.282	C301S/C308S/C326S	site-directed mutagenesis	Pyrococcus abyssi
2.1.1.282	V21C/C301S/C308S/K314C/C326S	site-directed mutagenesis	Pyrococcus abyssi

Metals/Ions

EC Number	Metals/Ions	Comment	Organism	Structure
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	Pyrococcus abyssi	-	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	Pyrococcus abyssi	Q9V2G1	-	-
2.1.1.282	Pyrococcus abyssi	Q9V2G1	-	-

Purification (Commentary)

EC Number	Purification (Comment)	Organism
2.1.1.228	recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and heparin affinity chromatography, and dialysis	Pyrococcus abyssi
2.1.1.282	recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity and heparin affinity chromatography, and dialysis	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	substrate-binding modes of PaTrm5a, and recognition of substrate analogues, overview	Pyrococcus abyssi	?	-	-
2.1.1.228	S-adenosyl-L-methionine + guanine37 in tRNAPhe	-	Pyrococcus abyssi	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	substrate-binding modes of PaTrm5a, and recognition of substrate analogues, 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	-	?

Subunits

EC Number	Subunits	Comment	Organism
2.1.1.228	?	x * 38500, recombinant N-terminally His6-tagged enzyme, SDS-PAGE	Pyrococcus abyssi
2.1.1.228	More	enzyme structure analysis, the 38.5-kDa protein is composed of three domains: D1 (M1-P60), D2 (N70-S162) and D3 (K163-S333) D1 and D2 are connected through an interdomain linker (M61-K69), overview	Pyrococcus abyssi
2.1.1.282	?	x * 38500, recombinant N-terminally His6-tagged enzyme, SDS-PAGE	Pyrococcus abyssi
2.1.1.282	More	enzyme structure analysis, he 38.5-kDa protein is composed of three domains: D1 (M1-P60), D2 (N70-S162) and D3 (K163-S333) D1 and D2 are connected through an interdomain linker (M61-K69), overview	Pyrococcus abyssi

Synonyms

EC Number	Synonyms	Comment	Organism
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
2.1.1.282	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

Cofactor

EC Number	Cofactor	Comment	Organism	Structure
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	evolution	during the evolutionary process, some euryarchaeota like Thermococcus and Pyrococcus preserved both the trm5 genes from the crenarchaeal origin as well as the native copy, but others apparently lost the latter. Phylogenetic distribution analyses of trm5 homologues in archaeal genomes allow the identification of three archaeal Trm5 (aTrm5) subfamilies: Trm5a, Trm5b, and Trm5c. Trm5b refers to the native form, while Trm5a refers to the crenarchaeal origin, and Trm5c to other members with divergent Trm5 sequences11. The three Trm5s differ substantially in primary sequences	Pyrococcus abyssi
2.1.1.228	metabolism	the enzyme is part of the The biosynthetic pathway of mimG in Pyrococcus abyssi, overview. In archaea, G37 hypermodification in tRNAPhe leads to wyosine derivatives. They are important in reading-frame maintenance during protein synthesis, while the absence of such modifications results in elevated error rates in +1 frame-shifting. Among the modification products, 7-methylwyosine (mimG) is perhaps the earliest and minimalist version of the wyosine derivatives unique to some archaea, and 4-demethylwyosine (imG-14), isowyosine (imG2) have also been identified as intermediates along the pathway. The first biosynthetic step of mimG is the formation of m1G37, catalysed by the S-adenosine-L-methionine (SAM)-dependent tRNA methyltransferase named Trm5, which belongs to class-I methyltransferases. The second step is the complex radical-mediated formation of imG-14, catalyzed by the radical SAM enzyme Taw1. The Trm5 enzyme from the archaeon Pyrococcus abyssi (PaTrm5a) also catalyzes the methylation of C7 on imG-14 to produce imG2 (EC 2.1.1.282), which is further methylated on the N4 position of the imidazo-purine ring by Taw3 to form mimG	Pyrococcus abyssi
2.1.1.228	additional information	enzyme structure comparisons	Pyrococcus abyssi
2.1.1.228	physiological function	tRNA methyltransferase Trm5 catalyses the transfer of a methyl group from S-adenosyl-L-methionine to G37 in eukaryotes and archaea. The N1-methylated guanosine is the product of the initial step of the wyosine hypermodification, which is essential for the maintenance of the reading frame during translation. As a unique member of this enzyme family, 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	Pyrococcus abyssi
2.1.1.282	evolution	during the evolutionary process, some euryarchaeota like Thermococcus and Pyrococcus preserved both the trm5 genes from the crenarchaeal origin as well as the native copy, but others apparently lost the latter. Phylogenetic distribution analyses of trm5 homologues in archaeal genomes allow the identification of three archaeal Trm5 (aTrm5) subfamilies: Trm5a, Trm5b, and Trm5c. Trm5b refers to the native form, while Trm5a refers to the crenarchaeal origin, and Trm5c to other members with divergent Trm5 sequences11. The three Trm5s differ substantially in primary sequences	Pyrococcus abyssi
2.1.1.282	metabolism	the enzyme is part of the biosynthetic pathway of mimG in Pyrococcus abyssi, overview. In archaea, G37 hypermodification in tRNAPhe leads to wyosine derivatives. They are important in reading-frame maintenance during protein synthesis, while the absence of such modifications results in elevated error rates in +1 frame-shifting. Among the modification products, 7-methylwyosine (mimG) is perhaps the earliest and minimalist version of the wyosine derivatives unique to some archaea, and 4-demethylwyosine (imG-14), isowyosine (imG2) have also been identified as intermediates along the pathway. The first biosynthetic step of mimG is the formation of m1G37, catalysed by the S-adenosine-L-methionine (SAM)-dependent tRNA methyltransferase named Trm5, which belongs to class-I methyltransferases. The second step is the complex radical-mediated formation of imG-14, catalyzed by the radical SAM enzyme Taw1. The Trm5 enzyme from the archaeon Pyrococcus abyssi (PaTrm5a) also catalyzes the methylation of C7 on imG-14 to produce imG2 (EC 2.1.1.282), which is further methylated on the N4 position of the imidazo-purine ring by Taw3 to form mimG	Pyrococcus abyssi
2.1.1.282	additional information	enzyme structure comparisons	Pyrococcus abyssi
2.1.1.282	physiological function	tRNA methyltransferase Trm5 catalyses the transfer of a methyl group from S-adenosyl-L-methionine to G37 in eukaryotes and archaea. The N1-methylated guanosine is the product of the initial step of the wyosine hypermodification, which is essential for the maintenance of the reading frame during translation. As a unique member of this enzyme family, 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	Pyrococcus abyssi

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Release 2023.1 (January 2023)