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Information on EC 5.4.99.12 - tRNA pseudouridine38-40 synthase and Organism(s) Escherichia coli and UniProt Accession P07649

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     5 Isomerases
         5.4 Intramolecular transferases
             5.4.99 Transferring other groups
                5.4.99.12 tRNA pseudouridine38-40 synthase
IUBMB Comments
The uridylate residues at positions 38, 39 and 40 of nearly all tRNAs are isomerized to pseudouridine. TruA specifically modifies uridines at positions 38, 39, and/or 40 in the anticodon stem loop of tRNAs with highly divergent sequences and structures .
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This record set is specific for:
Escherichia coli
UNIPROT: P07649
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Word Map
The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Reaction Schemes
tRNA uridine38-40
=
tRNA pseudouridine38-40
Synonyms
trna pseudouridine synthase i, mpus3p, pus3p, deg1p, pseudouridine synthase 3, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pseudouridine synthase I
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tRNA pseudouridine synthase I
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pseudouridine synthase
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pseudouridine synthase I
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tRNA pseudouridine synthase I
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
tRNA uridine38-40 = tRNA pseudouridine38-40
show the reaction diagram
tRNA uridine38-40 = tRNA pseudouridine38-40
show the reaction diagram
chemical evidence against a covalent cysteine intermediate in the rearrangement mechanism of uridine to pseudouridine
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SYSTEMATIC NAME
IUBMB Comments
tRNA-uridine38-40 uracil mutase
The uridylate residues at positions 38, 39 and 40 of nearly all tRNAs are isomerized to pseudouridine. TruA specifically modifies uridines at positions 38, 39, and/or 40 in the anticodon stem loop of tRNAs with highly divergent sequences and structures [1].
CAS REGISTRY NUMBER
COMMENTARY hide
430429-15-5
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61506-89-6
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
tRNA uridine38
tRNA pseudouridine38
show the reaction diagram
tRNALeu3 contains uridine at position 38. Wild-typeTruA pseudouridylates uridines at all three positions (38, 39 and 40) with efficiencies (kcat/KM) differing by less than 10fold, while R58A is inactive toward all three uridines. When flexibility of the anticodon stem loop is increased by mutating the two G:C base pairs in the stem of the anticodon stem loop of tRNALeu3 into A:U pairs, the kcat/KM increased 2fold. When flexibility is decreased by base-pairing the target U38 of tRNALeu3 with A32 instead of with U32, the kcat/KM decreases 10fold
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-
?
tRNA uridine38-40
tRNA pseudouridine38-40
show the reaction diagram
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
tRNA uridine40
tRNA pseudouridine40
show the reaction diagram
modified tRNALeu3 with uridine at position 40 instead of position 38. Wild-typeTruA pseudouridylates uridines at all three positions (38, 39 and 40) with efficiencies (kcat/KM) differing by less than 10fold, while R58A is inactive toward all three uridines
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-
?
Escherichia coli tRNAPhe uridine39
Escherichia coli tRNAPhe pseudouridine39
show the reaction diagram
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-
-
-
?
tRNA uridine38-40
tRNA pseudouridine38-40
show the reaction diagram
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purified tRNA pseudouridine synthase I modifies all of the hisT isoacceptors of tRNAHis, tRNATyr, and tRNALeu to products which are chromatographically indistinguishable from the respective wild-type species. These three groups of isoacceptors contain all the known topological sites for pseudouridine modification of residues 38,39, and 40
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?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
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tRNAPhe with uridine at position 39
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-
?
tRNAHis guanidine36 uridine38 cytidine39
RNAHis guanidine36 pseudouridine38 cytidine39
show the reaction diagram
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-
-
-
?
tRNAHis uridine38 cytidine39
RNAHis pseudouridine38 cytidine39
show the reaction diagram
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-
-
-
?
tRNAHis uridine38 uridine39
tRNAHis pseudouridine38 pseudouridine39
show the reaction diagram
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-
-
-
?
tRNALeu3
?
show the reaction diagram
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-
-
?
tRNALeu3 carrying uridine at position 38
tRNALeu3 carrying pseudouridine at position 38
show the reaction diagram
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-
-
?
tRNALeu3 carrying uridine at position 39
tRNALeu3 carrying pseudouridine at position 39
show the reaction diagram
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-
-
?
tRNALeu3 carrying uridine at position 40
tRNALeu3 carrying pseudouridine at position 40
show the reaction diagram
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-
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?
tRNAVal2a cytidine36 uridine38
tRNAVal2a cytidine36 pseudouridine38
show the reaction diagram
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poor substrate, tRNAVal2a is not a substrate
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?
additional information
?
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presence of a G36 residue modulates modification at position 38. In addition to local sequence effects, steady-state kinetic analyses suggest the existence of other recognition elements distinct from the immediate vicinity of modification
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
tRNA uridine38-40
tRNA pseudouridine38-40
show the reaction diagram
TruA specifically modifies uridines at positions 38, 39, and/or 40 of tRNAs with highly divergent sequences and structures
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-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
fluorouracil-substituted tRNA
causes a time-dependent inactivation of pseudouridine synthase I and forms a covalent complex with the enzyme that involves the fluorouracil-substituted UMP at position 39. Upon incubation of 100 nM pseudouridine synthase with 0.001 mM fluorouracil-substituted tRNA at 15°C prior to addition of substrate, there is a time-dependent inactivation of the enzyme with a half-life of 35 min
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5-fluorouracil tRNAPhe
time-dependent inactivation of pseudouridine synthase I and formation of a covalent complex with the enzyme that involves the 5-fluorouracil monophosphate at position 39
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Dithionitrobenzoate
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irreversibly inactivates
iodoacetate
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irreversibly inactivates
p-chloromercuribenzoate
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irreversibly inactivates
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00094 - 0.0018
tRNA uridine39
-
0.000086 - 0.00011
Escherichia coli tRNAPhe uridine39
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0.000086 - 0.00011
tRNA uridine39
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0.0000053
tRNAHis guanidine36 uridine38 cytidine39
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pH 8.0, 30°C
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0.0000804
tRNAHis uridine38 cytidine39
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pH 8.0, 30°C
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0.0000456
tRNAHis uridine38 uridine39
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pH 8.0, 30°C
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0.000669
tRNAVal2a cytidine36 uridine38
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pH 8.0, 30°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.18 - 0.73
tRNA uridine39
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0.0019
tRNAHis guanidine36 uridine38 cytidine39
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pH 8.0, 30°C
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0.0746
tRNAHis uridine38 cytidine39
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pH 8.0, 30°C
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0.144
tRNAHis uridine38 uridine39
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pH 8.0, 30°C
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0.0244
tRNAVal2a cytidine36 uridine38
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pH 8.0, 30°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
160
tRNA uridine38
pH 8.0, wild-type enzyme
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100 - 777
tRNA uridine39
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860
tRNA uridine40
pH 8.0, wild-type enzyme
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0.16
tRNALeu3
pH 8.0, 22°C
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0.2
tRNALeu3 carrying uridine at position 39
pH 8.0, 22°C
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0.86
tRNALeu3 carrying uridine at position 40
pH 8.0, 22°C
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0001
fluorouracil-substituted tRNA
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-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
-
assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
TruA utilizes the intrinsic flexibility of the ASL for site promiscuity and also to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation, thereby maintaining the balance between the flexibility and stability required for its biological function
physiological function
TruA utilizes the intrinsic flexibility of the anticodon stem loop for site promiscuity and also to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation, thereby maintaining the balance between the flexibility and stability required for its biological function
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
31000
x * 31000, SDS-PAGE
31000
-
x * 31000, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 31000, SDS-PAGE
?
-
x * 31000, SDS-PAGE
dimer
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapor diffusion method at room temperature. It is attempted to obtain structures of Escherichia coli TruA complexed with three Escherichia coli tRNAs representing all of the target sites: tRNALeu1 with uridine at 39, tRNALeu2 with uridine at 38 and 40, and tRNALeu3 with uridine at 38. These tRNAs are type II tRNAs with a 15 nucleotide variable loop. Three crystal forms are obtained from similar buffer conditions, containing the complex of the wild-type TruA and full-length tRNALeu1 in crystal I, and the complex of wild-type TruA and tRNALeu3 in crystal forms II and III. No crystals are obtained with tRNALeu2
crystal structures of TruA in complex with two different leucyl-tRNAs to 3.5-4.0 A resolution, in conjunction with functional assays and computer simulation. The structures capture three stages of the TruA-tRNA reaction, TruA utilizes the intrinsic flexibility of the anticodon stem loop for site promiscuity and also to select against intrinsically stable tRNAs to avoid their overstabilization through pseudouridylation, thereby maintaining the balance between the flexibility and stability required for its biological function
native protein, to 1.5 A resolution, and several derivatives. Structure reveals a dimeric protein that contains two positively charged, RNA-binding clefts along the surface of the protein. Each cleft contains a highly conserved aspartic acid located at its center. The structure suggests that a dimeric enzyme is required for binding transfer RNA and subsequent pseudouridine formation
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D60A
mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60E
mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60K
mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60N
mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60S
mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
R58A
wild-typeTruA pseudouridylates uridines at all three positions (38, 39 and 40) with efficiencies (kcat/KM) differing by less than 10fold, while R58A is inactive toward all three uridines. The wild-type and mutant enzymes have similar thermal stabilities based on identical tryptophan fluorescence curves over the range of melting temperatures, indicating that the R58A mutation does not drastically perturb the enzyme structure
C154A
C154S
C169A
C169S
C55A/C154A/C169A
D60A
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA
D60E
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA
D60K
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA
D60N
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA
D60S
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA
additional information
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
1 h, about 50% loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C or 2°C, 50% w/v glycerol, 50% loss of activity after 2 years
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
wild-type and mutant enzymes D60A, D60S, D60E, D60N, and D60K
isolation of virtually homogeneous tRNA pseudouridine synthase I from strains of Escherichia coli transformed with plasmid in which the production of tRNA pseudouridine synthase I is amplified 20fold
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
pseudouridine synthase I is cloned behind a T7 promoter and expressed in Escherichia coli to about 20% of total soluble proteins
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kammen, H.; Marvel, C.; Hardy, L.; Penhoet, E.
Purification structure and properties of Escherichia coli tRNA pseudouridine synthase I
J. Biol. Chem.
263
2255-2263
1988
Escherichia coli
Manually annotated by BRENDA team
Chihade, J.; Horne, D.
Single nucleotide modulation of uridine to pseudouridine rearrangement in transfer RNA catalyzed by pseudouridine synthase I
J. Mol. Recognit.
9
524-527
1996
Escherichia coli
Manually annotated by BRENDA team
Foster, P.G.; Huang, L.; Santi, D.; Stroud, R.
The structural basis of tRNA recognition and pseudouridine formation by pseudouridine synthase I
Nat. Struct. Biol.
7
23-27
2000
Escherichia coli
Manually annotated by BRENDA team
Gu, X.; Liu, Y.; Santi, D.V.
The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA
Proc. Natl. Acad. Sci. USA
96
14270-14275
1999
Saccharomyces cerevisiae, Escherichia coli (P07649)
Manually annotated by BRENDA team
Hur, S.; Stroud, R.M.
How U38, 39, and 40 of many tRNAs become the targets for pseudouridylation by TruA
Mol. Cell
26
189-203
2007
Escherichia coli, Escherichia coli (P07649)
Manually annotated by BRENDA team
Huang, L.; Pookanjanatavip, M.; Gu, X.; Santi, D.V.
A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst
Biochemistry
37
344-351
1998
Escherichia coli, Escherichia coli (P07649)
Manually annotated by BRENDA team
Zhao, X.; Horne, D.A.
The role of cysteine residues in the rearrangement of uridine to pseudouridine catalyzed by pseudouridine synthase I
J. Biol. Chem.
272
1950-1955
1997
Escherichia coli
Manually annotated by BRENDA team