Information on EC 2.1.1.260 - rRNA small subunit pseudouridine methyltransferase Nep1

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The expected taxonomic range for this enzyme is: Archaea, Eukaryota

EC NUMBER
COMMENTARY hide
2.1.1.260
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RECOMMENDED NAME
GeneOntology No.
rRNA small subunit pseudouridine methyltransferase Nep1
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA = S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
show the reaction diagram
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SYSTEMATIC NAME
IUBMB Comments
S-adenosyl-L-methionine:18S rRNA (pseudouridine1191-N1)-methyltransferase
This enzyme, which occurs in both prokaryotes and eukaryotes, recognizes specific pseudouridine residues (Psi) in small subunits of ribosomal RNA based on the local RNA structure. It recognizes Psi914 in 16S rRNA from the archaeon Methanocaldococcus jannaschii, Psi1191 in yeast 18S rRNA, and Psi1248 in human 18S rRNA.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
derived from the clinical isolate SC5314, gene NEP1
UniProt
Manually annotated by BRENDA team
derived from the clinical isolate SC5314, gene NEP1
UniProt
Manually annotated by BRENDA team
derived from the clinical isolate SC5314, gene NEP1
UniProt
Manually annotated by BRENDA team
derived from the clinical isolate SC5314, gene NEP1
UniProt
Manually annotated by BRENDA team
and isogenic strains, gene YLR186w or NEP1 or EMG1
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
S-adenosyl-L-methionine + pseudouridine1191 in yeast 18S rRNA
S-adenosyl-L-homocysteine + N1-methylpseudouridine1191 in yeast 18S rRNA
show the reaction diagram
additional information
?
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Nep1 target site identification by mass spectrometry, isotope-labeling, and fluorescence anisotropy measurements
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
S-adenosyl-L-methionine
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
sinefungin
sinefungin binds to Nep1 at a preformed binding site that is topologically equivalent to the cofactor binding site in other SPOUT-class methyltransferases
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant detagged wild-type and selenomethionine-labeled afNep1 dimer bound to S-adenosyl homocysteine, X-ray diffraction structure determination and analysis at 1.45-2.0 A resolution
Nep1 in its free form and bound to S-adenosylhomocysteine or the antibiotic and general methyltransferase inhibitor sinefungin, hanging drop vapour diffusion method, 10-15 mg/ml protein in 100 mM BisTris-propane buffer, pH 9.5, 6-10% PEG 400, 30-48% glycerol, and 0-400 mM trimethylamine-N-oxide, at 4°C, crystals of the selenomethionine containing protein in the presence or absence of a 3fold excess of S-adenosyl-L-homocysteine are grown under similar conditions at 16°C, X-ray diffraction structure determination and analysis at 2.2 A, 2.15 A, and 2.25 A resolution, respectively
recombinant detagged scNep1 dimer bound to S-adenosyl homocysteine and in complexes with RNA, i.e. one molecule and two molecules of cognate RNA, X-ray diffraction structure determination and analysis at 1.80-1.90 A and at 3.00 A resolution, respectively
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme partially from subcellular fractions by sucrose density gradient fractionation
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recombinant His-tagged Nep1 from Escherichia coli strain M15(pREP4) by nickel affinity chromatography
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recombinant His6-tagged Nep1 from Escherichia coli strain RosettaTM-DE3-pLysS by nickel affinity chromatography, cleavage of the tag by TEV protease, and two steps of gel filtration to over 99% purity
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recombinant N-terminally His6-tagged wild-type and D90G mutant Nep1 from Escherichia coli strain XL1-blue by nickel affinity chromatography and gel filtration
recombinant Nep1 by cation exchange chromatography and gel filtration
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recombinant Nep1p from Escherichia coli strain BL21(DE3) by ion exchange chromatography and gel filtration
recombinant wild-type and selenomethionine-labeled His6-tagged Nep1 from Escherichia coli strain RosettaTM-DE3-pLysS by nickel affinity chromatography, cleavage of the tag by TEV protease, and two steps of gel filtration to over 99% purity
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of His6-tagged Nep1 in Escherichia coli strain RosettaTM-DE3-pLysS
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expression of wild-type and selenomethionine-labeled His6-tagged Nep1 in Escherichia coli strain RosettaTM-DE3-pLysS
gene NEP1, DNA and amino acid sequence determination and analysis
gene NEP1, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain BL21(DE3)
gene NEP1, DNA and amino acid sequence determination and analysis, sequence comparisons, expression of the GFP-tagged ScNEP1 in enzyme-deficient Saccharomyces cerevisiae strain CEN.SR679 in the nucleus
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gene NEP1, expression of N-terminally His6-tagged wild-type and D90G mutant Nep1 in Escherichia coli strain XL1-blue
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gene NEP1, recombinaant expression of Nep1
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gene NEP1, recombinant expression of His-tagged Nep1 in Escherichia coli strain M15(pREP4)
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gene NEP1, stable expression of GFP-tagged wild-type and D86G mutant Nep1 in 293T cells under control of the tetracycline-inducible promoter, expression of N-terminally His6-tagged wild-type and D90G mutant Nep1 in Escherichia coli strain XL1-blue
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
S-adenosylmethionine suppression of Nep1, suppression of mutant ScDELTAsnr35 nep1-1ts is independent of psi1191 methylation
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D86G
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the mutation causes the Bowen-Conradi syndrome
D90G
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a mutation in yeast Nep1 equivalent to the Bowen-Conradi syndrome, BCS, mutation in the human Nep1. Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro
additional information