Literature summary for 2.1.1.220 extracted from

Wang, M.; Zhu, Y.; Wang, C.; Fan, X.; Jiang, X.; Ebrahimi, M.; Qiao, Z.; Niu, L.; Teng, M.; Li, X.
Crystal structure of the two-subunit tRNA m(1)A58 methyltransferase TRM6-TRM61 from Saccharomyces cerevisiae (2016), Sci. Rep., 6, 32562 .

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Cloned(Commentary)

Cloned (Comment)	Organism
genes GCD10 and GCD14, recombinant co-expression of wild-type and selenomethionine-labeled His-tagged subunits in Escherichia coli	Saccharomyces cerevisiae

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant wild-type and selenomethionine-labeled holoenzyme in apoform and complexed with S-adenosyl-L-methionine (SAM), mixing of 15 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 5 mM DTT, with a three-fold molecular excess of S-adenosyl-L-methionine, sitting drop vapour diffusion method, with the mother liquor containing 0.1 M HEPES, PH 7.5, 2% v/v 2-methyl-2,4-pentanediol, 10% w/v PEG 6000, 3 days X-ray diffraction structure determination and analysis	Saccharomyces cerevisiae

Crystallization (Comment)

Organism

purified recombinant wild-type and selenomethionine-labeled holoenzyme in apoform and complexed with S-adenosyl-L-methionine (SAM), mixing of 15 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 5 mM DTT, with a three-fold molecular excess of S-adenosyl-L-methionine, sitting drop vapour diffusion method, with the mother liquor containing 0.1 M HEPES, PH 7.5, 2% v/v 2-methyl-2,4-pentanediol, 10% w/v PEG 6000, 3 days X-ray diffraction structure determination and analysis

Saccharomyces cerevisiae

Protein Variants

Protein Variants	Comment	Organism
additional information	the human homologue of the yeast tRNA m1A58 methyltransferase is identified through amino acid sequence identity and complementation of the yeast temperature-sensitive TRM6 andTRM61 mutant phenotypes27. When co-expressed in yeast, the Homo sapiens TRM6-TRM61 catalyzes the in vitro methyl transfer reaction for both the yeast initiator tRNAi Met and human tRNA3Lys27	Saccharomyces cerevisiae

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	thermodynamics	Saccharomyces cerevisiae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
S-adenosyl-L-methionine + adenine58 in tRNA	Saccharomyces cerevisiae	-	S-adenosyl-L-homocysteine + N1-methyladenine58 in tRNA	-	?
S-adenosyl-L-methionine + adenine58 in tRNA	Saccharomyces cerevisiae ATCC 204508	-	S-adenosyl-L-homocysteine + N1-methyladenine58 in tRNA	-	?

Organism

Organism	UniProt	Comment	Textmining
Saccharomyces cerevisiae	P41814 AND P46959	subunits Trm6 and Trm61	-
Saccharomyces cerevisiae ATCC 204508	P41814 AND P46959	subunits Trm6 and Trm61	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and selenomethionine-labeled His-tagged subunits Trm6 and Trm61 from Escherichia coli by nickel affinity chromatography and gel filtration	Saccharomyces cerevisiae

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
S-adenosyl-L-methionine + adenine58 in tRNA	-	Saccharomyces cerevisiae	S-adenosyl-L-homocysteine + N1-methyladenine58 in tRNA	-	?
S-adenosyl-L-methionine + adenine58 in tRNA	-	Saccharomyces cerevisiae ATCC 204508	S-adenosyl-L-homocysteine + N1-methyladenine58 in tRNA	-	?

Subunits

Subunits	Comment	Organism
heterodimer	TRM6 and TRM61 form a compact complex via numerous hydrogen bonding and extensive hydrophobic interactions. The heterodimer interface of TRM6-TRM61 buries 3194 A2 of TRM6 and 3167 A2 of TRM61 solvent-accessible area, which represents about 17% and 16% of TRM6 and TRM61's total surface area, respectively. TRM6 mainly interacts with TRM61 through four major sites. Interaction analyses for sites A-C, detailed overview	Saccharomyces cerevisiae
heterotetramer	two TRM6-TRM61 heterodimers assemble as a heterotetramer. A symmetric unit of the TRM6-TRM61 crystal contains one molecule of TRM6 and one molecule of TRM61, forming a 1:1 heterodimer. TRM6 and TRM61 form a 2:2 tetrameric heterocomplex, displaying an omega shape. Two symmetry-related TRM6-TRM61 heterodimer come together to form a central beta-barrel structure that consists of beta13 (TRM6), loop beta13/beta14 (TRM6), beta12(TRM61) and loop beta13/beta14 (TRM61). The top of the barrel contains a hydrophobic core, formed by residues Tyr422 (TRM6), Pro431 (TRM6), Met253 (TRM61), His354 (TRM61), and Tyr357 (TRM61) The center of the barrel is filled with numerous hydrophilic side-chains, including residues Glu416 (TRM6), Arg418 (TRM6), Arg420 (TRM6), Glu255 (TRM61), Gln257 (TRM61) and Arg259 (TRM61). The bottom of the barrel consists of a cage of four tyrosine residues. Modelling of the heterotetramer interface of TRM6-TRM61, overview. A TRM6-TRM61 heterotetramer constitutes two L-shaped tRNA binding regions. Structure comparison to the enzyme from Homo sapiens	Saccharomyces cerevisiae

Synonyms

Synonyms	Comment	Organism
m1A58 tRNA methyl-transferase	-	Saccharomyces cerevisiae
TRM6-TRM61 holoenzyme	-	Saccharomyces cerevisiae
TrmI	-	Saccharomyces cerevisiae
tRNA m(1)A58 methyltransferase	-	Saccharomyces cerevisiae
two component m1A58 tRNA methyl-transferase	-	Saccharomyces cerevisiae

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Saccharomyces cerevisiae

Cofactor

Cofactor	Comment	Organism	Structure
S-adenosyl-L-methionine	SAM is situated in a cleft on the surface of the C-terminal Rossmann-fold domain of TRM61. The interaction between TRM61 and SAM can be divided into three parts in accordance to the moieties of SAM. For the adenine moiety, the side-chain atom OD1 of Asp168 makes a hydrogen bond with the nitrogen N6 of the adenine. Modeling of the SAM binding site and a possible adenine-binding pocket of TRM6-TRM61	Saccharomyces cerevisiae

General Information

General Information	Comment	Organism
evolution	TRM61 is the eukaryotic homologue of the bacterial and archaeal m1A58 tRNA methyl-transferase TrmI. Evolutionary relationship between TRM6 and TRM61, overview	Saccharomyces cerevisiae
additional information	two TRM6-TRM61 heterodimers assemble as a heterotetramer. Both TRM6 and TRM61 subunits comprise an N-terminal beta-barrel domain linked to a C-terminal Rossmann-fold domain. TRM61 functions as the catalytic subunit, containing a methyl donor (SAM) binding pocket. TRM6 diverges from TRM61, lacking the conserved motifs used for binding SAM. TRM6 cooperates with TRM61 forming an L-shaped tRNA binding regions. Target tRNA recognition and catalytic mechanism of the two component m1A58 tRNA methyl-transferase, overview	Saccharomyces cerevisiae
physiological function	the N1 methylation of adenine at position 58 (m1A58) of tRNA is an important post-transcriptional modification, which is vital for maintaining the stability of the initiator methionine tRNAiMet. Adenine at position 58 (A58) located in the T-loop is one of the most conserved nucleosides in tRNA. In eukaryotes, this modification is performed by the TRM6-TRM61 holoenzyme, molecular mechanism that underlies the cooperation of TRM6 and TRM61 in the methyl transfer reaction, overview	Saccharomyces cerevisiae