Information on EC 5.4.99.25 - tRNA pseudouridine55 synthase

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

EC NUMBER
COMMENTARY hide
5.4.99.25
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RECOMMENDED NAME
GeneOntology No.
tRNA pseudouridine55 synthase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
tRNA uridine55 = tRNA pseudouridine55
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
tRNA-uridine55 uracil mutase
Pseudouridine synthase TruB from Escherichia coli specifically modifies uridine55 in tRNA molecules [1]. The bifunctional archaeal enzyme also catalyses the pseudouridylation of uridine54 [6]. It is not known whether the enzyme from Escherichia coli can also act on position 54 in vitro, since this position is occupied in Escherichia coli tRNAs by thymine.
CAS REGISTRY NUMBER
COMMENTARY hide
430429-15-5
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61506-89-6
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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the finding that in archaea Cbf5 may function in tRNA uridine55 pseudouridylation as well as RNA-guided pseudouridylation of rRNA supports the idea that Cbf5 is a direct descendant of a primordial TruB/Pus4-like tRNA PSI synthase
malfunction
physiological function
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pseudouridine-55 synthase is responsible for modifying all tRNA molecules in the cell at the uridine55 position. TruB-effected pseudouridine55 modification of tRNA is not essential, but contributes to thermal stress tolerance in Escherichia. coli, possibly by optimizing the stability of the tRNA population at high temperatures
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
tRNA uridine55
tRNA pseudouridine55
show the reaction diagram
tRNA uridine55
tRNA pseudouridine552
show the reaction diagram
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substrate: Escherichia coli tRNAPhe
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-
?
tRNATrp uridine55
tRNATrp pseudouridine55
show the reaction diagram
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tRNATrp containing or lacking 3'-CCA. aCbf5 and aGar1 together can function as a tRNA Psi55 synthase in a guide RNA-independent manner. This activity is enhanced by aNop10, but not by L7Ae. The aCbf5 alone can also produce Psi55 in tRNAs that contain the canonical 3'-CCA sequence and this activity is stimulated by aGar1. tRNAs lacking 3'-CCA can be modified only by the aCbf5-aGar1 complex. The presence of conserved C (or U) and A at tRNA positions 56 and 58, respectively, is not essential for aCbf5-mediated Psi55 formation
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-
?
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
tRNA uridine55
tRNA pseudouridine55
show the reaction diagram
P48567
Pus4 catalyses the formation of pseudouridine55 in both mitochondrial and cytoplasmic tRNAs
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-
?
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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TruB is not inhibited by RNA containing 5-fluorouridine
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
aGar1 protein
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the aCbf5 alone can produce pseudouridine55 in tRNAs that contain the canonical 3’-CCA sequence. This activity is stimulated by aGar1
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aNop10 protein
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aCbf5 and aGar1 together can function as a tRNA pseudouridine55 synthase in a guide RNA-independent manner. This activity is enhanced by aNop10, but not by L7Ae
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protein aGar1
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aCbf5 alone can also produce Y55 in tRNAs that contain the canonical 3'-CCA sequence and this activity is stimulated by aGar1
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protein aNOP10
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additional information
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pfuCbf5 can modify tRNA uridine55, but not rRNA, in the absence of Gar1, Nop10 and L7Ae. The activity of pfuCbf5 toward tRNA uridine55 is enhanced in the presence of the accessory proteins, suggesting that a multi-protein Cbf5 complex may function in pseudouridine55 synthesis in Pyrococcus furiosus
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000124 - 0.78
tRNA uridine55
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additional information
additional information
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Km-values are determined for wild-type and mutant forms of yeast tRNAPhe
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.011 - 0.78
tRNA uridine55
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additional information
additional information
Escherichia coli
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turnover numbers are determined for wild-type and mutant forms of yeast tRNAPhe. The 7 base T-loop can be expanded or contracted by 1 base and still retains activity, albeit with a 30fold reduction in kcat
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7.43 - 1006
tRNA uridine32
4180
821.9 - 1469
tRNA uridine55
2626
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 80
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about 50% of maximal activity at 50°C and at 80°C, pseudouridine synthase Cbf5
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37300
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x * 37300, calculated from sequence
41500
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x * 41500, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 37300, calculated from sequence; x * 41500, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
atomic-resolution crystal structure, TruB bound to RNA at 1.85 A resolution
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the TruB crystallization construct (tTruB), differs from full-length Escherichia coli TruB in having an N-terminal His-tag followed by residue 10 of the full-length protein. tTruB(D48N) is cocrystallized with [5-fluorouridine]-RNA. Initially, crystals are obtained by seeding with fragments of tTruB–RNA cocrystals. The resulting crystals are crushed and used as seeds for six subsequent sequential rounds of seeding into tTruB(D48N)–RNA complex crystallization experiments
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vapor-phase diffusion method, crystal structure of TruB apoenzyme in the absence of RNA. Comparison of the TruB apoenzyme from Escherichia coli and the RNA-bound forms of Thermotoga maritima TruB provides insight into the structural basis for RNA recognition and specificity
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sitting drop vapor diffusion method, structure of pseudouridine55 tRNA in two substrate-free forms, at 1.9 A resolution
crystal structure is diffracted to a resolution of 1.7 A. The crystal belong to space group P2, with unit-cell parameters a = 37.65, b = 78.09, c = 56.33 A , c = 102.05
crystal structure of the Y67F mutant in complex with a 5-fluorouridine-tRNA at 2.4 A resolution
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vapor-phase diffusion method, crystal structure of TruB in complex with 17-base stem-loop of RNA. Comparison of the TruB apoenzyme from Escherichia coli and the RNA-bound form of Thermotoga maritima TruB provides insight into the structural basis for RNA recognition and specificity
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36
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Tm-value for mutant enzyme P20L is 35.9°C
38
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Tm-value for mutant enzyme P20G is 38.3°C
40
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Tm-value for wild-type enzyme is 40.2°C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
using Ni-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned in an Escherichia coli expression vector downstream of a His6-tag. The resulting recombinant protein Pus4 is expressed at high level
expressed in Escherichia coli as a His-tagged fusion protein
wild-type and mutant enzymes, expression in Escherichia coli. Wild-type enzyme can produce both pseudouridine54 and pseudouridine55 in Escherichia coli tRNAs
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C174A
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kcat is 1.3fold higher than wild-type value, KM is 1.1fold lower than wild-type value
C193V
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kcat is 1.5fold higher than wild-type value, KM is 1.5fold higher than wild-type value
C58A
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kcat is 2.2fold higher than wild-type value, KM is 1.2fold higher than wild-type value
C58A/C174A/C193A
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kcat is 1.1fold lower than wild-type value, KM is 1.2fold lower than wild-type value
D48N
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inactive mutant enzyme
D90A
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dissociation constant: 0.4 microM (wild-type: 0.7 microM), rate of pseudouridine formation compared to wild-type: 500fold decreased, rate of tRNA binding (kapp2): 2/sec (wild-type: 4.2/sec)
D90E
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dissociation constant: 0.4 microM (wild-type: 0.7 microM), rate of pseudouridine formation compared to wild-type: 30fold decreased, rate of tRNA binding (kapp2): 1.5/sec (wild-type: 4.2/sec)
D90N
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dissociation constant: 0.4 microM (wild-type: 0.7 microM), rate of pseudouridine formation compared to wild-type: 50fold decreased, rate of tRNA binding (kapp2): 1.9/sec (wild-type: 4.2/sec)
K19M
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kcat is 8fold lower than wild-type value. KM-value is 11fold higher than wild-type value
K19R
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kcat is 10fold lower than wild-type value. KM-value is 6fold higher than wild-type value
P20G
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kcat is 4.8fold lower than wild-type value. KM-value is 2.2fold higher than wild-type value
P20L
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kcat is 2.1fold lower than wild-type value. KM-value is 3.5fold higher than wild-type value
R181A
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dissociation constant: 2 microM (wild-type: 0.7 microM), rate of pseudouridine formation compared to wild-type: more than 20000fold decreased, rate of tRNA binding (kapp2): 4/sec (wild-type: 4.2/sec)
R181K
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dissociation constant: 0.7 microM (wild-type: 0.7 microM), rate of pseudouridine formation compared to wild-type: 2500fold decreased, rate of tRNA binding (kapp2): 1.9/sec (wild-type: 4.2/sec)
R181M
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dissociation constant: 0.5 microM (wild-type: 0.7 microM), rate of pseudouridine formation compared to wild-type: more than 20000fold decreased, rate of tRNA binding (kapp2): 3/sec (wild-type: 4.2/sec)
C106A/C109A
decrease in tRNA pseudouridine54 synthase activity, no decrease in tRNA pseudouridine55 synthase activity
D275A
the mutant shows no tRNA pseudouridine54 synthase activity and no tRNA pseudouridine55 synthase activity
D277A
decrease in tRNA pseudouridine54 synthase activity and low decrease in tRNA pseudouridine55 synthase activity
H376A/R377A
decrease in tRNA pseudouridine54 synthase activity and in tRNA pseudouridine55 synthase activity
I412A
decrease in tRNA pseudouridine54 synthase activity and in tRNA pseudouridine55 synthase activity
K413A
decrease in tRNA pseudouridine54 synthase activity and in tRNA pseudouridine55 synthase activity
L440A
the mutant shows no tRNA pseudouridine54 synthase activity and no tRNA pseudouridine55 synthase activity
R273A
decrease in tRNA pseudouridine54 synthase activity and in tRNA pseudouridine55 synthase activity
Y339A
the mutant shows no tRNA pseudouridine54 synthase activity and no tRNA pseudouridine55 synthase activity
D275A
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the mutant shows no tRNA pseudouridine54 synthase activity and no tRNA pseudouridine55 synthase activity
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D277A
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decrease in tRNA pseudouridine54 synthase activity and low decrease in tRNA pseudouridine55 synthase activity
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I412A
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decrease in tRNA pseudouridine54 synthase activity and in tRNA pseudouridine55 synthase activity
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R273A
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decrease in tRNA pseudouridine54 synthase activity and in tRNA pseudouridine55 synthase activity
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Y339A
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the mutant shows no tRNA pseudouridine54 synthase activity and no tRNA pseudouridine55 synthase activity
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K53A
substitution K53A or R202A in aCBF5 impairs both the tRNA:pseudouridine55-synthase and the RNA-guided RNA:pseudouridine-synthase activities
R202A
substitution K53A or R202A in aCBF5 impairs both the tRNA:pseudouridine55-synthase and the RNA-guided RNA:pseudouridine-synthase activities
Y67F
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no activity with the natural RNA substrate, efficient formation of 5-fluoro-6-hydroxypseudouridine55 from 5-fluorouridine55
Y67L
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no activity with the natural RNA substrate, efficient formation of 5-fluoro-6-hydroxypseudouridine55 from 5-fluorouridine55
additional information
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