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Literature summary for 2.1.1.202 extracted from

  • Liu, R.; Long, T.; Li, J.; Li, H.; Wang, E.
    Structural basis for substrate binding and catalytic mechanism of a human RNA m5C methyltransferase NSun6 (2017), Nucleic Acids Res., 45, 6684-6697 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.1.1.202

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His6-tagged wild-type enzyme HNSun6 and mutant enzymes Homo sapiens

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant His6-tagged enzyme, hanging drop vapor diffusion method, mixing of 0.001 ml of 4.8 mg/ml protein in 20 mM Tris-HCl, pH 7.0, 300 mM NaCl, 5 mM MgCl2, and 5 mM DTT, with 0.001 ml of reservoir solution consisting of 2% dioxane, 0.1 M bicine, pH 8.6, 18% w/v PEG 8000, and 10 mM yttrium(III) chloride hexahydrate, 16°C. For the hNSun6/tRNA complex, solutions are prepared with 0.075 mM MhNSun6 and 0.075 mM MtRNA, for the hNSun6/tRNA/SAM and hNSun6/tRNA/SFG complexes, hNSun6 is mixed with tRNA at a 1:1 molar ratio, adding either 1 mM SAM or SFG, crystals are obtained by mixing 0.001 ml of this solution with 0.001 ml of reservoir solution containing 0.1 M SPG, pH 8.0-10.0, 24-28% w/v PEG 1500, and 50 mM NaF, X-ray diffraction structure determination and analysis. Of different human tRNACys and tRNAThr isoacceptors and their variants, only tRNACys(GCA)-G2A:C71U can be cocrystallized with hNSun6, molecular replacement using the MTase domain structure of Pyrococcus horikoshii PH0851 (PDB ID: 2YXL) and the PUA domain structure of Pyrococcus horikoshii archaeosine tRNA-guanine transglycosylase (PDB ID: 1J2B) as starting models Homo sapiens

Protein Variants

Protein Variants Comment Organism
C326A site-directed mutagenesis, tRNA binding structure compared to wild-type enzyme, the C326A mutation hampers the breakdown of the covalent intermediates Homo sapiens
D266A site-directed mutagenesis, the mutant shows highly increased binding affinity for SAM and highly reduced methylation activity Homo sapiens
D293A site-directed mutagenesis, the mutation leads to a loss of SAM binding capability and a loss of methylation activity to tRNACys Homo sapiens
D323A site-directed mutagenesis, the mutant shows highly increased binding affinity for SAM and highly reduced methylation activity Homo sapiens
F141A site-directed mutagenesis, the mutant shows activity similar to wild-type enzyme Homo sapiens
K159A site-directed mutagenesis, the mutant shows moderately decreased activity compared to wild-type enzyme Homo sapiens
K159A/R181A site-directed mutagenesis, the mutant shows highly decreased activity compared to wild-type enzyme Homo sapiens
K160A site-directed mutagenesis, the mutant shows moderately decreased activity compared to wild-type enzyme Homo sapiens
K160A/R181A site-directed mutagenesis, the mutant shows highly decreased activity compared to wild-type enzyme Homo sapiens
K248A site-directed mutagenesis, the mutant shows 5fold increased binding affinity for SAM Homo sapiens
K271A site-directed mutagenesis, the mutant shows highly increased binding affinity for SAM and highly reduced methylation activity Homo sapiens
additional information generation of trm4a and trm4a/trm4b deletion mutants, tRNA methylome of wild-type, trm4aDELTA, trm4bDELTA, and trm4aDELTA/trm4bDELTA as determined by genome-wide high-throughput bisulfite sequencing, overview Schizosaccharomyces pombe
additional information generation of trm4b and trm4a/trm4b deletion mutants, tRNA methylome of wild-type, trm4aDELTA, trm4bDELTA, and trm4aDELTA/trm4bDELTA as determined by genome-wide high-throughput bisulfite sequencing, overview Schizosaccharomyces pombe
R181A site-directed mutagenesis, the mutant shows highly decreased activity compared to wild-type enzyme Homo sapiens

Inhibitors

Inhibitors Comment Organism Structure
additional information structure-based drug design toward RNA:m5C methyltransferase-related diseases Homo sapiens

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Homo sapiens
Mg2+ required Schizosaccharomyces pombe

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
S-adenosyl-L-methionine + cytosine34 in tRNA Schizosaccharomyces pombe
-
 S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine34 in tRNA Schizosaccharomyces pombe ATCC 24843
-
 S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine34 in tRNA Schizosaccharomyces pombe 972
-
 S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine34 in tRNAPro(CGG) Schizosaccharomyces pombe
-
 S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine34 in tRNAPro(CGG) Schizosaccharomyces pombe ATCC 24843
-
 S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine34 in tRNAPro(CGG) Schizosaccharomyces pombe 972
-
 S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNA Schizosaccharomyces pombe
-
 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNA Schizosaccharomyces pombe ATCC 24843
-
 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNA Schizosaccharomyces pombe 972
-
 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNALeu(CAA) Schizosaccharomyces pombe
-
 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNALeu(CAA)
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNALeu(CAA) Schizosaccharomyces pombe ATCC 24843
-
 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNALeu(CAA)
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNALeu(CAA) Schizosaccharomyces pombe 972
-
 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNALeu(CAA)
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNA Schizosaccharomyces pombe
-
 S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNA Schizosaccharomyces pombe ATCC 24843
-
 S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNA Schizosaccharomyces pombe 972
-
 S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNAPro(CGG) Schizosaccharomyces pombe
-
 S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNAPro(CGG) Schizosaccharomyces pombe ATCC 24843
-
 S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNAPro(CGG) Schizosaccharomyces pombe 972
-
 S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNA Schizosaccharomyces pombe
-
 S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNA Schizosaccharomyces pombe ATCC 24843
-
 S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNA Schizosaccharomyces pombe 972
-
 S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNAPro(CGG) Schizosaccharomyces pombe
-
 S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNAPro(CGG) Schizosaccharomyces pombe ATCC 24843
-
 S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNAPro(CGG) Schizosaccharomyces pombe 972
-
 S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine72 in tRNACys(GCA) Homo sapiens
-
 S-adenosyl-L-homocysteine + 5-methylcytosine72 in tRNACys(GCA)
-
 ?

Organism

Organism UniProt Comment Textmining
Homo sapiens Q8TEA1
-
-
Schizosaccharomyces pombe O13935
-
-
Schizosaccharomyces pombe Q9HGQ2
-
-
Schizosaccharomyces pombe 972 O13935
-
-
Schizosaccharomyces pombe 972 Q9HGQ2
-
-
Schizosaccharomyces pombe ATCC 24843 O13935
-
-
Schizosaccharomyces pombe ATCC 24843 Q9HGQ2
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged enzyme HNSun6, hNSun6 and hNSun6-C326A-RNA complexes by nickel affinity chromatography Homo sapiens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information C34 methylation depends on Trm4a in both tRNALeuCAA and tRNAProCGG. Substrate specificity, comparison to trm4b, overview Schizosaccharomyces pombe ?
-
-
additional information the CCA end is precisely recognized by hNSun6 primarily through the PUA domain, overview. The main chains of the residues (Arg126, Pro206 and Asp209) recognizes C74. Recognition for C75 comes from the main chain residues (Lys192 and Gly193) and the side chain residues (Lys192 and Asp209). The base moiety of C75 is stacked with the Tyr131 residue. Recognition for A76 is achieved mostly by the main chain of His129 and the side chain of Lys 192. Hydrophobic interactions with ambient aa residues, including Cys120, facilitate localization of the A76 base moiety. The discriminator base U73 binds to the RRM motif. C72 is recognized by the catalytic core. Multiple roles of the PUA domain in tRNA binding Homo sapiens ?
-
-
additional information Trm4b is active on the wild-type tRNA, its activity is slightly enhanced on C34A and completely abrogated on C49A, indicating that Trm4b has in vitro activity on tRNAProCGG and is specific for C49, which is in agreement with in vivo methylation data. Substrate specificity, comparison to trm4a, overview Schizosaccharomyces pombe ?
-
-
additional information Trm4b is active on the wild-type tRNA, its activity is slightly enhanced on C34A and completely abrogated on C49A, indicating that Trm4b has in vitro activity on tRNAProCGG and is specific for C49, which is in agreement with in vivo methylation data. Substrate specificity, comparison to trm4a, overview Schizosaccharomyces pombe ATCC 24843 ?
-
-
additional information C34 methylation depends on Trm4a in both tRNALeuCAA and tRNAProCGG. Substrate specificity, comparison to trm4b, overview Schizosaccharomyces pombe ATCC 24843 ?
-
-
additional information Trm4b is active on the wild-type tRNA, its activity is slightly enhanced on C34A and completely abrogated on C49A, indicating that Trm4b has in vitro activity on tRNAProCGG and is specific for C49, which is in agreement with in vivo methylation data. Substrate specificity, comparison to trm4a, overview Schizosaccharomyces pombe 972 ?
-
-
additional information C34 methylation depends on Trm4a in both tRNALeuCAA and tRNAProCGG. Substrate specificity, comparison to trm4b, overview Schizosaccharomyces pombe 972 ?
-
-
S-adenosyl-L-methionine + cytosine34 in tRNA
-
 Schizosaccharomyces pombe S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine34 in tRNA
-
 Schizosaccharomyces pombe ATCC 24843 S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine34 in tRNA
-
 Schizosaccharomyces pombe 972 S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine34 in tRNAPro(CGG)
-
 Schizosaccharomyces pombe S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine34 in tRNAPro(CGG)
-
 Schizosaccharomyces pombe ATCC 24843 S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine34 in tRNAPro(CGG)
-
 Schizosaccharomyces pombe 972 S-adenosyl-L-homocysteine + 5-methylcytosine34 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNA
-
 Schizosaccharomyces pombe S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNA
-
 Schizosaccharomyces pombe ATCC 24843 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNA
-
 Schizosaccharomyces pombe 972 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNALeu(CAA)
-
 Schizosaccharomyces pombe S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNALeu(CAA)
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNALeu(CAA)
-
 Schizosaccharomyces pombe ATCC 24843 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNALeu(CAA)
-
 ?
S-adenosyl-L-methionine + cytosine48 in tRNALeu(CAA)
-
 Schizosaccharomyces pombe 972 S-adenosyl-L-homocysteine + 5-methylcytosine48 in tRNALeu(CAA)
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNA
-
 Schizosaccharomyces pombe S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNA
-
 Schizosaccharomyces pombe ATCC 24843 S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNA
-
 Schizosaccharomyces pombe 972 S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNAPro(CGG)
-
 Schizosaccharomyces pombe S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNAPro(CGG)
-
 Schizosaccharomyces pombe ATCC 24843 S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine49 in tRNAPro(CGG)
-
 Schizosaccharomyces pombe 972 S-adenosyl-L-homocysteine + 5-methylcytosine49 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNA
-
 Schizosaccharomyces pombe S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNA
-
 Schizosaccharomyces pombe ATCC 24843 S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNA
-
 Schizosaccharomyces pombe 972 S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNA
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNAPro(CGG)
-
 Schizosaccharomyces pombe S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNAPro(CGG)
-
 Schizosaccharomyces pombe ATCC 24843 S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine50 in tRNAPro(CGG)
-
 Schizosaccharomyces pombe 972 S-adenosyl-L-homocysteine + 5-methylcytosine50 in tRNAPro(CGG)
-
 ?
S-adenosyl-L-methionine + cytosine72 in tRNACys(GCA)
-
 Homo sapiens S-adenosyl-L-homocysteine + 5-methylcytosine72 in tRNACys(GCA)
-
 ?
S-adenosyl-L-methionine + cytosine72 in tRNACys(GCA)-G2A:C71U
-
 Homo sapiens S-adenosyl-L-homocysteine + 5-methylcytosine72 in tRNACys(GCA)-G2A:C71U
-
 ?

Synonyms

Synonyms Comment Organism
HNSun6
-
 Homo sapiens
NSUN6
-
 Homo sapiens
RNA:m5C methyltransferase
-
 Homo sapiens
RNA:m5C MTase
-
 Homo sapiens
TRM4A
-
 Schizosaccharomyces pombe
TRM4B
-
 Schizosaccharomyces pombe

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Schizosaccharomyces pombe
37
-
 assay at Homo sapiens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Homo sapiens
7.5
-
 assay at Schizosaccharomyces pombe

Cofactor

Cofactor Comment Organism Structure
S-adenosyl-L-methionine
-
 Schizosaccharomyces pombe
S-adenosyl-L-methionine cofactor interacting residues in hNSun6 are mainly composed of conserved residues Leu241, Cys242, Pro325 and Leu354. In addition, the side chain of residue Asp293 recognizes the N6 of the adenine ring. Asp293 is conserved as Asp or Glu in the RNA:m5C MTase family Homo sapiens

General Information

General Information Comment Organism
evolution phylogenetic tree of Trm4/ NSun2 homologues, overview. Enzyme Trm4a is responsible for in vivo methylation of C34 and C48 methylation, whereas Trm4b methylates C49 and C50, tRNAProCGG is the only tRNA methylated by both Trm4a and Trm4b. Schizosaccharomyces pombe shows an unusual separation of activities of the NSun2/Trm4 enzymes that are united in a single enzyme in other eukaryotes like humans, mice and Saccharomyces cerevisiae Schizosaccharomyces pombe
evolution phylogenetic tree of Trm4/ NSun2 homologues, overview. In contrast to most other organisms, fission yeast Schizosaccharomyces pombe carries two Trm4/NSun2 homologues, Trm4a (SPAC17D4.04) and Trm4b (SPAC23C4.17). Enzyme Trm4a is responsible for in vivo methylation of C34 and C48 methylation, whereas Trm4b methylates C49 and C50, tRNAProCGG is the only tRNA methylated by both Trm4a and Trm4b. Schizosaccharomyces pombe shows an unusual separation of activities of the NSun2/Trm4 enzymes that are united in a single enzyme in other eukaryotes like humans, mice and Saccharomyces cerevisiae Schizosaccharomyces pombe
malfunction the absence of Trm4a, but not Trm4b, causes a mild resistance of Schizosaccharomyces pombe to calcium chloride. Absence of Trm4a, but not Trm4b, causes a mild resistance of Schizosaccharomyces pombe to calcium chloride. In trm4aDELTA mutants, the C34 methylation level drops to background levels (8%), whereas C49 methylation remains at 82%. m5C34 is at 94% in trm4bDELTA mutant, and m5C49 drops to 9%, thus confirming that Trm4a is responsible for m5C34 and Trm4b for m5C49 on tRNAProCGG in vivo Schizosaccharomyces pombe
malfunction the absence of Trm4a, but not Trm4b, causes a mild resistance of Schizosaccharomyces pombe to calcium chloride. In trm4aDELTA mutants, the C34 methylation level drops to background levels (8%), whereas C49 methylation remains at 82%. m5C34 is at 94% in trm4bDELTA mutant, and m5C49 drops to 9%, thus confirming that Trm4a is responsible for m5C34 and Trm4b for m5C49 on tRNAProCGG in vivo Schizosaccharomyces pombe
additional information analysis of the catalytic mechanism of the RNA:m5C methyltransferase family, structure-function analysis of RNA:m5C methyltransferases, overview. Lys248, Asp323, Cys326 and Cys373 are the residues at the active site of hNSun6, they are strictly conserved in NSun6s and in the entire RNA:m5CMTase family, suggesting their conserved roles in catalysis. Multiple roles of Lys248 and Asp323. Motif IVCys (C326) plays a role in product release. In the hNSun6/tRNA complex, instead of nucleotide flipping, the acceptor region of tRNA undergoes a complicated conformational reconstitution for access to hNSun6 Homo sapiens
physiological function enzymes of the cytosine-5 RNA methyltransferase Trm4/NSun2 family methylate tRNAs at C48 and C49 in multiple tRNAs, as well as C34 and C40 in selected tRNAs. Trm4a is responsible for all C48 methylation, which lies in the tRNA variable loop, as well as for C34 in tRNALeuCAA and tRNAProCGG, which are at the anticodon wobble position. Trm4a displays intron-dependent methylation of C34 Schizosaccharomyces pombe
physiological function Enzymes of the cytosine-5 RNA methyltransferase Trm4/NSun2 family methylate tRNAs at C48 and C49 in multiple tRNAs, as well as C34 and C40 in selected tRNAs. Trm4b methylates C49 and C50, which both lie in the TPsiC-stem. Trm4b activity of methylation of C34 is independent of the intron Schizosaccharomyces pombe