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Information on EC 2.1.1.217 - tRNA (adenine22-N1)-methyltransferase and Organism(s) Bacillus subtilis and UniProt Accession P54471
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2.1.1.217 tRNA (adenine22-N1)-methyltransferaseIUBMB Comments
The enzyme specifically methylates adenine22 in tRNA.
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Word Map
- 2.1.1.217
- mtases
- methyltransferases
- adomet
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rna-binding
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groove
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sam-dependent
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class-i
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ligand-free
The taxonomic range for the selected organisms is: Bacillus subtilis
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Reaction Schemes
+
adenine22 in tRNA
=
+
N1-methyladenine22 in tRNA
Synonyms
bstrmk, class i mtase, sp1610, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:tRNA (adenine22-N1)-methyltransferase
The enzyme specifically methylates adenine22 in tRNA.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + adenine22 in Bacillus subtilis tRNA(Ser)
S-adenosyl-L-homocysteine + N1-methyladenine22 in Bacillus subtilis tRNA(Ser)
crude Escherichia coli tRNA is used, since it does not contain m1A22
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-
?
S-adenosyl-L-methionine + adenine22 in tRNA
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA
S-adenosyl-L-methionine + adenine22 in tRNASer
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNASer
S-adenosyl-L-methionine + adenine22 in tRNATyr
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNATyr
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?
S-adenosyl-L-methionine + adenine22 in tRNA
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA
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-
-
?
S-adenosyl-L-methionine + adenine22 in tRNA
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA
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-
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-
?
S-adenosyl-L-methionine + adenine22 in tRNA
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA
a methyl group at position N1 prevents Watson-Crick-type base pairing by adenosine and is therefore important for regulation of structure and stability of tRNA molecules
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-
?
S-adenosyl-L-methionine + adenine22 in tRNA
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA
crude Escherichia coli tRNA is used, since it does not contain m1A22
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-
?
S-adenosyl-L-methionine + adenine22 in tRNASer
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNASer
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-
-
?
S-adenosyl-L-methionine + adenine22 in tRNASer
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNASer
interaction of BstRNASer with BsTrmK/SAH is deciphered by NMR. The tRNASer produced in Escherichia coli is less efficiently modified than the unmodified tRNASer
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?
additional information
?
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the methyltransferase TrmK (BsTrmK) is responsible for the formation of m1A22 in tRNA. BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of Bacillus subtilis bearing an A22. In addition to a non-Watson-Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. Measurements of the MTase activity using 32P-radiolabelled Bacillus subtilis tRNAs. Interaction between BsTrmK and the cofactor product S-adenosyl-L-homocysteine (SAH) is enthalpy-driven with a single binding site and a dissociation constant (KD) of 0.0017 mM, residues R9, L10, G77, D78, A94 and G95 are involved in SAH binding
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additional information
?
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the methyltransferase TrmK (BsTrmK) is responsible for the formation of m1A22 in tRNA. BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of Bacillus subtilis bearing an A22. In addition to a non-Watson-Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. Measurements of the MTase activity using 32P-radiolabelled Bacillus subtilis tRNAs. Interaction between BsTrmK and the cofactor product S-adenosyl-L-homocysteine (SAH) is enthalpy-driven with a single binding site and a dissociation constant (KD) of 0.0017 mM, residues R9, L10, G77, D78, A94 and G95 are involved in SAH binding
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + adenine22 in tRNA
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA
S-adenosyl-L-methionine + adenine22 in tRNASer
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNASer
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-
-
?
S-adenosyl-L-methionine + adenine22 in tRNA
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA
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-
-
?
S-adenosyl-L-methionine + adenine22 in tRNA
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA
-
-
-
-
?
S-adenosyl-L-methionine + adenine22 in tRNA
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA
a methyl group at position N1 prevents Watson-Crick-type base pairing by adenosine and is therefore important for regulation of structure and stability of tRNA molecules
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-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
S-adenosyl-L-methionine
SAM, enzyme binding structure analysis, overview. SAM is modelled into the active site of BsTrmK, guided by the crystal structure of SAM-bound TrmK from Streptomyces pneumoniae (PDB ID 3KU1). The SAM cofactor is bound at the centre of the catalytic domain in a pocket with residues harbouring negative electrostatic potentials conserved amongst COG2384 proteins. The methyl group is pointing towards a region of positively charged and well conserved residues, likely important for tRNA binding
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
DTT
the higher oligomeric states of BsTrmK are formed via disulphide bonds involving the two cysteines in BsTrmK sequence at positions 35 and 152. Such bonds can be broken by addition of a reducing-agent, and addition of DTT to the MTase reaction buffer results in a dramatic increase of the enzymatic activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.000042
adenine22 in tRNASer
recombinant enzyme, pH 8.0, 37°C, in vitro activity
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0.000079
adenine22 in tRNASer
recombinant enzyme, pH 8.0, 37°C, in vivo activity
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additional information
additional information
Michaelis-Menten kinetics, steady-state kinetic analysis of BsTrmK
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additional information
additional information
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Michaelis-Menten kinetics, steady-state kinetic analysis of BsTrmK
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
Bacillus subtilis TrmK belongs to the COG2384 (cluster of orthologous groups). The members of this family are found in Gram-negative and Gram-positive bacteria. Their sequences are well-conserved in many bacterial pathogens
additional information
additional information
NMR chemical shift mapping is used to get insight on the protein-RNA recognition mode
additional information
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NMR chemical shift mapping is used to get insight on the protein-RNA recognition mode
additional information
the crystal structure of BsTrmK shows an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. Docking of BstRNASer to BsTrmK in complex with its methyldonor cofactor S-adenosyl-L-methionine (SAM) by NMR chemical shift mapping, modelling, overview. Both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson-Crick R13-A22 base-pair positioning the A22 N1-atom close to the SAM methyl group. BsTrmK requires the intact three-dimensional structure of tRNA to catalyse m1A22 formation
additional information
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the crystal structure of BsTrmK shows an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. Docking of BstRNASer to BsTrmK in complex with its methyldonor cofactor S-adenosyl-L-methionine (SAM) by NMR chemical shift mapping, modelling, overview. Both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson-Crick R13-A22 base-pair positioning the A22 N1-atom close to the SAM methyl group. BsTrmK requires the intact three-dimensional structure of tRNA to catalyse m1A22 formation
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
BsTrmK is active as a monomer, the higher oligomeric states of BsTrmK are formed via disulphide bonds involving the two cysteines in BsTrmK sequence at positions 35 and 152. Such bonds can be broken by addition of a reducing-agent, and addition of DTT to the MTase reaction buffer results in a dramatic increase of the enzymatic activity
additional information
additional information
backbone 1H, 15N and 13C chemical shift assignments of TrmK from Bacillus subtilis obtained by heteronuclear multidimensional NMR spectroscopy as well as its secondary structure in solution, overview
additional information
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backbone 1H, 15N and 13C chemical shift assignments of TrmK from Bacillus subtilis obtained by heteronuclear multidimensional NMR spectroscopy as well as its secondary structure in solution, overview
additional information
BsTrmK consists of an N-terminal Class I MTase domain linked to a C-terminal coiled-coil domain, structure comparisons, overview
additional information
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BsTrmK consists of an N-terminal Class I MTase domain linked to a C-terminal coiled-coil domain, structure comparisons, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method, mixing of 0.0016 ml of 3 mg/ml protein in 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 2% glycerol, with crystallization solution containing 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate, pH 5.0, 30% w/v PEG 4000, and 8% 2-methyl-2,4-pentanediol, and equuilibration against 0.1 ml reservoir solution, microseeding, 4°C, X-ray diffraction structure determination and analysis, molecular replacement using structures of sp1610 (Streptococcus pneumoniae orthologue of BsTrmK, PDB code 3KR9), LMOf2365 1472 (Listeria Monocytogens, PDB code 3GNL) and of SAG1203 (Streptococcus agalactiae, PDB code: 3LEC) as templates, modelling
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C35S/C152S
site-directed mutagenesis, since wild-type TrmK is prone to aggregation through cysteine oxidation, a protein variant with cysteine to serine mutations (i.e. C35S and C152S) is produced, which shows abolished protein aggregation and retains full enzymatic activity
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant N-terminally His6-tagged mutant enzyme C35S/C152S from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by thrombin, followed by ultrafiltration and gel filtration
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene trmK, recombinant overexpression of N-terminally His6-tagged mutant enzyme C35S/C152S in Escherichia coli strain BL21(DE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Roovers, M.; Kaminska, K.H.; Tkaczuk, K.L.; Gigot, D.; Droogmans, L.; Bujnicki, J.M.
The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK)
Nucleic Acids Res.
36
3252-3262
2008
Bacillus subtilis (P54471), Bacillus subtilis
Degut, C.; Barraud, P.; Larue, V.; Tisne, C.
Backbone resonance assignments of the m1A22 tRNA methyltransferase TrmK from Bacillus subtilis
Biomol. NMR Assign.
10
253-257
2016
Bacillus subtilis (P54471), Bacillus subtilis, Bacillus subtilis 168 (P54471)
Degut, C.; Roovers, M.; Barraud, P.; Brachet, F.; Feller, A.; Larue, V.; Al Refaii, A.; Caillet, J.; Droogmans, L.; Tisne, C.
Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK insights into tRNA recognition
Nucleic Acids Res.
47
4736-4750
2019
Bacillus subtilis (P54471), Bacillus subtilis, Bacillus subtilis 168 (P54471), no activity in Escherichia coli
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