Literature summary for 2.1.1.217 extracted from

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 (2019), Nucleic Acids Res., 47, 4736-4750 .

Search for more literature for 2.1.1.217

Activating Compound

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(Commentary)

Cloned (Comment)	Organism
gene trmK, recombinant expression of His-tagged enzyme	Bacillus subtilis

Crystallization (Commentary)

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

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 [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Bacillus subtilis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
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

Organism	UniProt	Comment	Textmining
Bacillus subtilis	P54471	-	-
Bacillus subtilis 168	P54471	-	-
no activity in Escherichia coli	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme by nickel affinity chromatography	Bacillus subtilis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

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 [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Bacillus subtilis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Bacillus subtilis

Cofactor

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

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