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Literature summary for 2.1.1.207 extracted from
- The tRNA recognition mechanism of the minimalist SPOUT methyltransferase, TrmL (2013), Nucleic Acids Res., 41, 7828-7842.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene trmL, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Escherichia coli |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| purified enzyme TrmL in apoform and in complex with S-adenosyl-homocysteine, X-ray diffraction structure determination and analysis at 2.0 A resolution, molecular replacement using the structure of Haemophilus influenzae Yibk, PDB ID 1J85 as starting search model, modeling | Escherichia coli |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| K42E | site-directed mutagenesis, inactive mutant | Escherichia coli |
| K81E | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
| additional information | construction of gene trmL deletion mutants | Escherichia coli |
| R104E | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Escherichia coli |
| R129E | site-directed mutagenesis, inactive mutant | Escherichia coli |
| R20E | site-directed mutagenesis, inactive mutant | Escherichia coli |
| R28E | site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme | Escherichia coli |
| R43E | site-directed mutagenesis, inactive mutant | Escherichia coli |
| R45E | site-directed mutagenesis, inactive mutant | Escherichia coli |
| R46E | site-directed mutagenesis, inactive mutant | Escherichia coli |
| R59E | site-directed mutagenesis, inactive mutant | Escherichia coli |
| R64E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Escherichia coli |
| R74E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Escherichia coli |
| Y142A | site-directed mutagenesis, the mutant is a monomer in contrast to the wild-type enzyme. Mutant Y142A fails to form a complex with tRNA | Escherichia coli |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | steady-state kinetic parameters of EcTrmL with modified tRNALeu substrates | Escherichia coli |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Escherichia coli |  |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 19800 | - |
2 * 19800, SDS-PAGE and gel filtration, the overall structure of an EcTrmL monomer subunit is composed of six beta-strands and six alpha-helices, in the order beta1-alpha1-beta2-alpha2-alpha3-beta3-alpha4-beta4-beta5-alpha5-beta6-alpha6. EcTrmL dimer formation is essential for tRNA recognition. The residue Y142 is critical for maintaining the dimeric form of EcTrmL, which is consistent with its central position at the interface | Escherichia coli |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | - |
gene trmL | - |
| Escherichia coli MT102 | - |
gene trmL | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Escherichia coli |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | the enzyme TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to tRNALeuCAA and tRNALeuUAA isoacceptors without the involvement of other tRNA-binding proteins. EcTrmL alone can efficiently methylate both tRNALeuCAA and tRNALeuUAA isoacceptors by using in vivo-purified tRNA substrates with certain modifications, but not unmodified tRNALeu | Escherichia coli | ? | - |
? | |
| additional information | the enzyme TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to tRNALeuCAA and tRNALeuUAA isoacceptors without the involvement of other tRNA-binding proteins. EcTrmL alone can efficiently methylate both tRNALeuCAA and tRNALeuUAA isoacceptors by using in vivo-purified tRNA substrates with certain modifications, but not unmodified tRNALeu | Escherichia coli MT102 | ? | - |
? | |
| S-adenosyl-L-methionine + cytidine34 in tRNALeuCAA | - |
Escherichia coli | S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuCAA | - |
? | |
| S-adenosyl-L-methionine + cytidine34 in tRNALeuCAA | - |
Escherichia coli MT102 | S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuCAA | - |
? | |
| S-adenosyl-L-methionine + cytidine34 in tRNALeuUAA | - |
Escherichia coli | S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuUAA | - |
? | |
| S-adenosyl-L-methionine + cytidine34 in tRNALeuUAA | - |
Escherichia coli MT102 | S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuUAA | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| homodimer | 2 * 19800, SDS-PAGE and gel filtration, the overall structure of an EcTrmL monomer subunit is composed of six beta-strands and six alpha-helices, in the order beta1-alpha1-beta2-alpha2-alpha3-beta3-alpha4-beta4-beta5-alpha5-beta6-alpha6. EcTrmL dimer formation is essential for tRNA recognition. The residue Y142 is critical for maintaining the dimeric form of EcTrmL, which is consistent with its central position at the interface | Escherichia coli |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| Trml | - |
Escherichia coli |
| tRNA (Um34/Cm34) methyltransferase | - |
Escherichia coli |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Escherichia coli |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 8 | - |
assay at | Escherichia coli |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| S-adenosyl-L-methionine | analysis of the structure of the cofactor binding pocket | Escherichia coli | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | TrmL is a member of the SPOUT superfamily. TrmH, TrmJ and TrmL belong to the SpoU family, and TrmD belongs to the TrmD family. Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Domain architectures of SPOUT tRNA MTases and the sequence alignment of TrmLs, overview. TrmL enzymes are widely distributed throughout the bacterial kingdom | Escherichia coli |
| additional information | the enzyme TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition. Analysis of the structure of the active site. EcTrmL dimer formation is essential for tRNA recognition. The residue Y142 is critical for maintaining the dimeric form of EcTrmL, which is consistent with its central position at the interface | Escherichia coli |
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