Information on EC 2.1.1.74 - methylenetetrahydrofolate-tRNA-(uracil54-C5)-methyltransferase [NAD(P)H-oxidizing]

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718903, 719972

folate/FAD-dependent tRNA T54 methyltransferase

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methylenetetrahydrofolate-transfer ribonucleate uracil 5-methyltransferase,

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TRMFO

6 entries

tRNA (m5U54)-methyltransferase

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tRNA:m5U-54 MTase

P39815

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676202

5,10-methylenetetrahydrofolate + uracil54 in tRNA + NAD(P)H + H+ = tetrahydrofolate + 5-methyluracil54 in tRNA + NAD(P)+

methyl group transfer

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reductive methylation

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5,10-methylenetetrahydrofolate:tRNA (uracil54-C5)-methyltransferase

A flavoprotein (FAD). Up to 25% of the bases in mature tRNA are post-translationally modified or hypermodified. One almost universal post-translational modification is the conversion of U54 into ribothymidine in the TPsiC loop, and this modification is found in most species studied to date [2]. Unlike this enzyme, which uses 5,10-methylenetetrahydrofolate and NAD(P)H to supply the atoms for methylation of U54, EC 2.1.1.35, tRNA (uracil54-C5)-methyltransferase, uses S-adenosyl-L-methionine.

56831-74-4

74665-78-4 deleted

5,10-methylenetetrahydrofolate + tRNA containing uridine at position 54 + FADH2

tetrahydrofolate + tRNA containing ribothymidine at position 54 + FAD

3 entries

5,10-methylenetetrahydrofolate + tRNA UpsiC + FADH2

tetrahydrofolate + tRNA TpsiC + FAD

17 entries

5,10-methylenetetrahydrofolate + tRNA UpsiC + NADH

tetrahydrofolate + tRNA TpsiC + NAD+

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-

?

5,10-methylenetetrahydrofolate + uracil54 in tRNA + FADH2

tetrahydrofolate + 5-methyluracil54 in tRNA + FAD

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756874

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?

N5,N10-methylenetetrahydrofolate + tRNA containing uridine at position 54 + FADH2

tetrahydrofolate + tRNA containing ribothymidine at position 54 + FAD

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-

?

S-adenosyl-L-methionine + tRNAPhe containing uridine at position 54

S-adenosyl-L-homocysteine + tRNAPhe containing ribothymidine at position 54

S-adenosyl-L-methionine + tRNAPhe mutant A58G containing uridine at position 54

S-adenosyl-L-homocysteine + tRNAPhe mutant A58G containing ribothymidine at position 54

S-adenosyl-L-methionine + tRNAPhe mutant A58U containing uridine at position 54

S-adenosyl-L-homocysteine + tRNAPhe mutant A58U containing ribothymidine at position 54

S-adenosyl-L-methionine + tRNAPhe mutant G57A containing uridine at position 54

S-adenosyl-L-homocysteine + tRNAPhe mutant G57A containing ribothymidine at position 54

S-adenosyl-L-methionine + tRNAPhe nucleotides 18-76 containing uridine at position 54

S-adenosyl-L-homocysteine + tRNAPhe nucleotides 18-76 containing ribothymidine at position 54

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-

?

S-adenosyl-L-methionine + tRNAPhe nucleotides 44-76 containing uridine at position 54

S-adenosyl-L-homocysteine + tRNAPhe nucleotides 44-76 containing ribothymidine at position 54

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-

?

S-adenosyl-L-methionine + tRNAPhe with deletions of aminoacyl stem and D-arm containing uridine at position 54

S-adenosyl-L-homocysteine + tRNAPhe with deletions of aminoacyl stem and D-arm containing ribothymidine at position 54

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-

?

S-adenosyl-L-methionine + tRNAPhe with deletions of aminoacyl stem containing uridine at position 54

S-adenosyl-L-homocysteine + tRNAPhe with deletions of aminoacyl stem containing ribothymidine at position 54

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-

?

S-adenosyl-L-methionine + tRNAPhe with deletions of anticodon arm containing uridine at position 54

S-adenosyl-L-homocysteine + tRNAPhe with deletions of anticodon armand D-arm containing ribothymidine at position 54

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-

?

S-adenosyl-L-methionine + tRNAPhe with deletions of D-arm containing uridine at position 54

S-adenosyl-L-homocysteine + tRNAPhe with deletions of D-arm containing ribothymidine at position 54

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-

?

additional information

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4 entries

5,10-methylenetetrahydrofolate + tRNA containing uridine at position 54 + FADH2

tetrahydrofolate + tRNA containing ribothymidine at position 54 + FAD

5,10-methylenetetrahydrofolate + tRNA UpsiC + FADH2

tetrahydrofolate + tRNA TpsiC + FAD

FAD

FADH2

NADH

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is used in the assay as electron donor

NADPH

0.5

5,10-methylenetetrahydrofolate

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485583

0.0011

tRNAPhe containing uridine at position 54

pH not specified in the publication, 60°C

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0.0022

tRNAPhe mutant A58G containing uridine at position 54

pH not specified in the publication, 60°C

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0.0013

tRNAPhe mutant A58U containing uridine at position 54

pH not specified in the publication, 60°C

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0.0021

tRNAPhe mutant G57A containing uridine at position 54

pH not specified in the publication, 60°C

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0.0045

tRNAPhe nucleotides 18-76 containing uridine at position 54

pH not specified in the publication, 60°C

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0.0026

tRNAPhe nucleotides 44-76 containing uridine at position 54

pH not specified in the publication, 60°C

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0.0019

tRNAPhe with deletions of aminoacyl stem and D-arm containing uridine at position 54

pH not specified in the publication, 60°C

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0.0013

tRNAPhe with deletions of aminoacyl stem containing uridine at position 54

pH not specified in the publication, 60°C

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0.0012

tRNAPhe with deletions of anticodon arm containing uridine at position 54

pH not specified in the publication, 60°C

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0.0015

tRNAPhe with deletions of D-arm containing uridine at position 54

pH not specified in the publication, 60°C

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0.1

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485582, 485583

7.5

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assay at

718903, 720967

8

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assay at

9

25

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assay at

37

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assay at

60

assay at

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-

-

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Clostridium acidi-urici

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Clostridium botulinum

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Geobacillus stearothermophilus

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485581, 485582, 485583

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-

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Mycobacterium leprae

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Mycobacterium tuberculosis

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Thermus thermophilus DSM 579

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no activity in Micrococcus luteus

strains ATCC 4698 and ATCC12698, formerly Micrococcus lysodeikticus

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-

-

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UniProt

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UniProt

Treponema palladium

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cytoplasm

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5737

485581

physiological function

a gene disruption strain does show any growth retardation at 70°C, but shows a slow-growth phenotype at 50°C with an increase in 2'-O-methylguanosine at position 18 and decrease in N1-methyladenosine at position 58 in the tRNA mixture from the mutant strain at 50°C. The protein synthesis activity of the mutant strain is inferior to that of the wild-type strain at 50°C

756874

3 entries

Thermus thermophilus HB8

33600

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untagged protein

47900

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His-tagged fusion protein

free TrmFO or bound to tetrahydrofolate-bound or glutathione, addition of GSH greatly improves the quality of the crystals, X-ray diffraction structure determinationand analysis at 2.1 A, 1.6 A, and 1.05 A resolutions, respectively

purified recombinant enzyme, from 25% v/v PEG 4000, 100 mM NaCl and sodium citrate buffer, pH 5.0, at 291 K using the hanging-drop vapor-diffusion method in 0.0016 ml with 8 mg/ml protein, 7 days, X-ray diffraction structure determination and analysis at 2.6 A resolution

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C193A

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mutant is nearly as active as the wild-type enzyme

C226A

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mutant loses both the tRNA methylation activity and the capacity to form a covalent complex with the 5-FU-mini-RNA

C53A

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mutant is inactive but like the wild-type enzyme, mutant C53A is capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. Mutation of Cys-53 changes the accessibility of the FAD-binding site and impairs the conformational stability of TrmFO

C53A/C226A

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as for the single C226A mutant, no protein-RNA covalent complex is detectable with the double mutant

R312G

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mutant isolated after cloning is found to be defective in tRNA methylation, with an activity corresponding only to 40% that of the wild-type enzyme

S54A

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mutant is nearly as active as the wild-type enzyme

C223A

site-directed mutagenesis, almost inactive mutant

C51A

site-directed mutagenesis, almost inactive mutant

E341A

site-directed mutagenesis, the active mutant shows a decrease in both FAD binding and methylation activity compared to the wild-type enzyme

H308A

site-directed mutagenesis, the mutant shows 57% of wild-type enzyme activity compared to the wild-type enzyme

K282A

site-directed mutagenesis, almost inactive mutant

K287A

site-directed mutagenesis, almost inactive mutant

K409A

site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme

K410A

site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme

N310A

site-directed mutagenesis, the mutant shows 23% of wild-type enzyme activity compared to the wild-type enzyme

R97A

site-directed mutagenesis, the mutant shows decreased activity compared to the wild-type enzyme

W283A

site-directed mutagenesis, the mutant shows slightly decreased activity

enzyme requires FAD+ for stability

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485582

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485582, 485583

purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by gel filtration

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recombinant enzyme from Escherichia coli strain BL21(DE3) by anion and cation exchange chromatography and gel filtration to over 95% purity

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recombinant enzyme from Escherichia coli to homogeneity

using Ni-NTA chromatography

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718903

expressed in Escherichia coli as a His-tagged fusion protein

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718903

expression in Escherichia coli

overexpression in Escherichia coli strain BL21(DE3)

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the encoded recombinant protein contains tightly bound flavin and is active in Escherichia coli mutant lacking m5U-54 in tRNAs and in vitro using T7 tRNA transcript as substrate

P39815

676202

untagged N-terminus and C-terminus (His)6-tagged TrmFO from Bacillus subtilis is expressed in Escherichia coli. The tag does not significantly alter the expression level, flavin content, activity and secondary structure of the protein

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