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Literature summary for 2.1.1.200 extracted from

  • Liu, R.J.; Long, T.; Zhou, M.; Zhou, X.L.; Wang, E.D.
    tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily (2015), Nucleic Acids Res., 43, 7489-7503.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of C-terminally His6-tagged wild-type enzyme and deletion mutants in Escherichia coli strain BL21(DE3) Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant C-terminally His6-tagged wild-type enzyme, hanging drop vapour diffusion method, mixing of 10 mg/ml of protein 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2, and 2 mM S-adenosyl-L-homocysteine, with 3.6 M NaCl and 0.1 M HEPES, pH 8.2, 20°C, 2 months, X-ray diffraction structure determination and analysis, full-length EcTrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations in the crystal structure, molecular replacement Escherichia coli

Protein Variants

Protein Variants Comment Organism
E225A site-directed mutagenesis, inactive mutant Escherichia coli
F199A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
G231A site-directed mutagenesis, inactive mutant Escherichia coli
I228A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
L229A site-directed mutagenesis, inactive mutant Escherichia coli
additional information generation of several partial deletion mutants of TrmJ, including single site mutants, DELTA2 (deleted residues 171-172), DELTA4 (deleted residues 171-174), DELTA7 (deleted residues 169-175), DELTA10 (deleted residues 167-176), DELTA12 (deleted residues 166-177), NTD (residues 1-170) and CTD (residues 171-246) Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.00067
-
nucleoside32 in EctRNAfMet1(CAU) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli
0.00081
-
nucleoside32 in EctRNAfMet2(CAU) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli
0.00094
-
nucleoside32 in EctRNATrp1(CCA) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli
0.00579
-
nucleoside32 in EctRNAGln2(CUG) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli
0.00988
-
nucleoside32 in EctRNAGln1(UUG) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli
0.01182
-
nucleoside32 in EctRNASer1(UGA) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
additional information Escherichia coli bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2'-O modification at ribose 32. All six Escherichia coli tRNAs with 2'-O-methylated nucleosides at position 32 are substrates of EcTrmJ. The elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and this process is regulated by the flexible hinge region that connects these two domains ?
-
?
additional information Escherichia coli K-12 MG1655 bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2'-O modification at ribose 32. All six Escherichia coli tRNAs with 2'-O-methylated nucleosides at position 32 are substrates of EcTrmJ. The elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and this process is regulated by the flexible hinge region that connects these two domains ?
-
?
S-adenosyl-L-methionine + nucleoside32 in tRNA Escherichia coli
-
S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in tRNA
-
?
S-adenosyl-L-methionine + nucleoside32 in tRNA Escherichia coli K-12 MG1655
-
S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in tRNA
-
?

Organism

Organism UniProt Comment Textmining
Escherichia coli P0AE01
-
-
Escherichia coli K-12 MG1655 P0AE01
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant C-terminally His6-tagged wild-type enzyme and deletion mutants 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 bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2'-O modification at ribose 32. All six Escherichia coli tRNAs with 2'-O-methylated nucleosides at position 32 are substrates of EcTrmJ. The elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and this process is regulated by the flexible hinge region that connects these two domains Escherichia coli ?
-
?
additional information bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2'-O modification at ribose 32. All six Escherichia coli tRNAs with 2'-O-methylated nucleosides at position 32 are substrates of EcTrmJ. The elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and this process is regulated by the flexible hinge region that connects these two domains Escherichia coli K-12 MG1655 ?
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNAfMet1(CAU)
-
Escherichia coli S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAfMet1(CAU)
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNAfMet1(CAU)
-
Escherichia coli K-12 MG1655 S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAfMet1(CAU)
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNAfMet2(CAU)
-
Escherichia coli S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAfMet2(CAU)
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNAfMet2(CAU)
-
Escherichia coli K-12 MG1655 S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAfMet2(CAU)
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNAGln1(UUG)
-
Escherichia coli S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAGln1(UUG)
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNAGln1(UUG)
-
Escherichia coli K-12 MG1655 S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAGln1(UUG)
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNAGln2(CUG)
-
Escherichia coli S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNAGln2(CUG)
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNASer1(UGA)
-
Escherichia coli S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNASer1(UGA)
-
?
S-adenosyl-L-methionine + nucleoside32 in EctRNATrp1(CCA)
-
Escherichia coli S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in EctRNATrp1(CCA)
-
?
S-adenosyl-L-methionine + nucleoside32 in tRNA
-
Escherichia coli S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in tRNA
-
?
S-adenosyl-L-methionine + nucleoside32 in tRNA
-
Escherichia coli K-12 MG1655 S-adenosyl-L-homocysteine + 2'-O-methylnucleoside32 in tRNA
-
?

Subunits

Subunits Comment Organism
More the dimerization of C-terminal domain is important for the catalytic activity of EcTrmJ Escherichia coli

Synonyms

Synonyms Comment Organism
TrmJ
-
Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.02
-
nucleoside32 in EctRNAGln1(UUG) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli
0.0218
-
nucleoside32 in EctRNATrp1(CCA) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli
0.0235
-
nucleoside32 in EctRNAGln2(CUG) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli
0.0237
-
nucleoside32 in EctRNAfMet2(CAU) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli
0.0253
-
nucleoside32 in EctRNAfMet1(CAU) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli
0.0383
-
nucleoside32 in EctRNASer1(UGA) pH 9.0, 37°C, recombinant wild-type enzyme Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
9
-
assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
S-adenosyl-L-methionine
-
Escherichia coli

General Information

General Information Comment Organism
evolution TrmJ proteins from the SPOUT methyltransferase superfamily are tRNA Xm32 modification enzymes that occur in bacteria and archaea. Unlike archaeal TrmJ, bacterial TrmJ require full-length tRNA molecules as substrates Escherichia coli
physiological function methylation of ribose moieties in tRNA is frequent, especially at position 32 where it is commonplace in all three domains of life Escherichia coli