Cloned (Comment) | Organism |
---|---|
gene trm5a, recombinant expression of His6-tagged wild-type and mutant enzymes | Pyrococcus abyssi |
Crystallization (Comment) | Organism |
---|---|
analysis of enzyme complex structures of Trm5b (MjTrm5b, PDB IDs 2YX1 and 3AY0) from Methanococcus jannaschii | Methanocaldococcus jannaschii |
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 |
Protein Variants | Comment | Organism |
---|---|---|
D243A | site-directed mutagenesis, substrate binding compared to wild-type enzyme | Pyrococcus abyssi |
E213A | site-directed mutagenesis, substrate binding compared to wild-type enzyme | Pyrococcus abyssi |
H128A | site-directed mutagenesis, substrate binding compared to wild-type enzyme | Pyrococcus abyssi |
R133A | site-directed mutagenesis, substrate binding compared to wild-type enzyme | Pyrococcus abyssi |
R135A | site-directed mutagenesis, substrate binding compared to wild-type enzyme | Pyrococcus abyssi |
R174A | site-directed mutagenesis, substrate binding compared to wild-type enzyme | Pyrococcus abyssi |
Y318A | site-directed mutagenesis, substrate binding compared to wild-type enzyme | Pyrococcus abyssi |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Methanocaldococcus jannaschii | |
Mg2+ | required | Pyrococcus abyssi |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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) | ? | - |
- |
|
S-adenosyl-L-methionine + guanine37 in tRNAPhe | Methanocaldococcus jannaschii | - |
S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe | - |
? | |
S-adenosyl-L-methionine + guanine37 in tRNAPhe | Pyrococcus abyssi | - |
S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe | - |
? | |
S-adenosyl-L-methionine + guanine37 in tRNAPhe | Methanocaldococcus jannaschii DSM 2661 | - |
S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Methanocaldococcus jannaschii | Q58293 | - |
- |
Methanocaldococcus jannaschii DSM 2661 | Q58293 | - |
- |
Pyrococcus abyssi | Q9V2G1 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes | Pyrococcus abyssi |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | ? | - |
- |
|
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 | ? | - |
- |
|
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 | ? | - |
- |
|
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 | ? | - |
- |
|
S-adenosyl-L-methionine + guanine37 in tRNAPhe | - |
Methanocaldococcus jannaschii | S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe | - |
? | |
S-adenosyl-L-methionine + guanine37 in tRNAPhe | - |
Pyrococcus abyssi | S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe | - |
? | |
S-adenosyl-L-methionine + guanine37 in tRNAPhe | - |
Methanocaldococcus jannaschii DSM 2661 | S-adenosyl-L-homocysteine + N1-methylguanine37 in tRNAPhe | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | substrate interactions with the catalytic region in the PaTrm5a-tRNAPhe-SAH ternary complex, overview | Pyrococcus abyssi |
Synonyms | Comment | Organism |
---|---|---|
MjTrm5b | - |
Methanocaldococcus jannaschii |
More | see also EC 2.1.1.282 | Pyrococcus abyssi |
PaTrm5a | - |
Pyrococcus abyssi |
TAW22 | - |
Pyrococcus abyssi |
Trm5a | - |
Pyrococcus abyssi |
trm5b | - |
Methanocaldococcus jannaschii |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Methanocaldococcus jannaschii |
37 | - |
assay at | Pyrococcus abyssi |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Methanocaldococcus jannaschii |
8 | - |
assay at | Pyrococcus abyssi |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
S-adenosyl-L-methionine | - |
Methanocaldococcus jannaschii | |
S-adenosyl-L-methionine | - |
Pyrococcus abyssi |
General Information | Comment | Organism |
---|---|---|
malfunction | deletion of the D1 domain greatly reduces the affinity and activity of PaTrm5a toward its RNA substrate | Pyrococcus abyssi |
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 |
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 |
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 |