Activating Compound | Comment | Organism | Structure |
---|---|---|---|
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 | Bacillus subtilis |
Cloned (Comment) | Organism |
---|---|
gene trmK, recombinant expression of His-tagged enzyme | Bacillus subtilis |
Crystallization (Comment) | Organism |
---|---|
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 | Bacillus subtilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics, steady-state kinetic analysis of BsTrmK | Bacillus subtilis | |
0.000042 | - |
adenine22 in tRNASer | recombinant enzyme, pH 8.0, 37°C, in vitro activity | Bacillus subtilis | |
0.000079 | - |
adenine22 in tRNASer | recombinant enzyme, pH 8.0, 37°C, in vivo activity | Bacillus subtilis |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Bacillus subtilis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
S-adenosyl-L-methionine + adenine22 in tRNA | Bacillus subtilis | - |
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA | - |
? | |
S-adenosyl-L-methionine + adenine22 in tRNA | Bacillus subtilis 168 | - |
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA | - |
? | |
S-adenosyl-L-methionine + adenine22 in tRNASer | Bacillus subtilis | - |
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNASer | - |
? | |
S-adenosyl-L-methionine + adenine22 in tRNASer | Bacillus subtilis 168 | - |
S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNASer | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bacillus subtilis | P54471 | - |
- |
Bacillus subtilis 168 | P54471 | - |
- |
no activity in Escherichia coli | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged enzyme by nickel affinity chromatography | Bacillus subtilis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | 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 | Bacillus subtilis | ? | - |
- |
|
additional information | 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 | Bacillus subtilis 168 | ? | - |
- |
|
S-adenosyl-L-methionine + adenine22 in tRNA | - |
Bacillus subtilis | S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA | - |
? | |
S-adenosyl-L-methionine + adenine22 in tRNA | - |
Bacillus subtilis 168 | S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNA | - |
? | |
S-adenosyl-L-methionine + adenine22 in tRNASer | - |
Bacillus subtilis | S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNASer | - |
? | |
S-adenosyl-L-methionine + adenine22 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 | Bacillus subtilis | S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNASer | - |
? | |
S-adenosyl-L-methionine + adenine22 in tRNASer | - |
Bacillus subtilis 168 | S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNASer | - |
? | |
S-adenosyl-L-methionine + adenine22 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 | Bacillus subtilis 168 | S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNASer | - |
? | |
S-adenosyl-L-methionine + adenine22 in tRNATyr | - |
Bacillus subtilis | S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNATyr | - |
? | |
S-adenosyl-L-methionine + adenine22 in tRNATyr | - |
Bacillus subtilis 168 | S-adenosyl-L-homocysteine + N1-methyladenine22 in tRNATyr | - |
? |
Subunits | Comment | Organism |
---|---|---|
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 | Bacillus subtilis |
More | BsTrmK consists of an N-terminal Class I MTase domain linked to a C-terminal coiled-coil domain, structure comparisons, overview | Bacillus subtilis |
Synonyms | Comment | Organism |
---|---|---|
BsTrmK | - |
Bacillus subtilis |
Class I MTase | - |
Bacillus subtilis |
m1A22 tRNA methyltransferase | - |
Bacillus subtilis |
TrmK | - |
Bacillus subtilis |
yqfN | - |
Bacillus subtilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Bacillus subtilis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Bacillus subtilis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
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 | Bacillus subtilis |
General Information | Comment | Organism |
---|---|---|
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 | Bacillus subtilis |