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Information on EC 5.4.99.25 - tRNA pseudouridine55 synthase and Organism(s) Escherichia coli and UniProt Accession P60340
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5.4.99.25 tRNA pseudouridine55 synthaseIUBMB Comments
Pseudouridine synthase TruB from Escherichia coli specifically modifies uridine55 in tRNA molecules . The bifunctional archaeal enzyme also catalyses the pseudouridylation of uridine54 . 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.
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Word Map
- 5.4.99.25
- synthases
-
pseudouridylation
-
anticodon
-
rna-guided
- 5-fluorouridine
-
srnps
-
thumb
- snornas
-
eukaryal
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
pus10, acbf5, psi synthase, pseudouridine synthase trub, pseudouridine 55 synthase, trna pseudouridine synthase, psi55s, ynl292w, trna:pseudouridine-55 synthase, rna pseudouridine synthase trub, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
tRNA uridine55 = tRNA pseudouridine55
RluA does not require thiol(ate) groups to effect catalysis
tRNA uridine55 = tRNA pseudouridine55
this enzyme recognizes the preformed three-dimensional structure of the T loop primarily through shape complementarity. It accesses its substrate uridyl residue by flipping out the nucleotide and disrupts the tertiary structure of tRNA
tRNA uridine55 = tRNA pseudouridine55
this enzyme undergoes significant conformational changes on binding to its substrate. These conformational changes include the ordering of the thumb loop, which binds right into the RNA hairpin loop, and a 10° hinge movement of the C-terminal domain. TruB recognizes its RNA substrate through a combination of rigid docking and induced fit, with TruB first rigidly binding to its target and then maximizing the interaction by induced fit
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
430429-15-5
-
61506-89-6
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
tRNA uridine55
tRNA pseudouridine552
substrate: Escherichia coli tRNAPhe
-
-
?
tRNA uridine55
tRNA pseudouridine55
substrate: Escherichia coli tRNAPhe
-
-
?
tRNA uridine55
tRNA pseudouridine55
the recombinant protein is specific for uridine55 in tRNA transcripts and reacts neither at other sites for PSI in such transcripts nor with transcripts of 16S or 23S ribosomal RNA or subfragments. Uridine54, uridine32 and uridine39 are not converted to pseudouridine. Stoichiometric formation of psi occurs with no requirement for an external source of energy, indicating that PSI synthesis is thermodynamically favored
-
-
?
tRNA uridine55
tRNA pseudouridine55
-
activity is determined with yeast tRNAPhe (wild-type and mutants). A 17 base oligoribonucleotide analog of the T-arm is equivalent to intact native tRNA as a substrate for pseudouridine 55 synthase. The structures and activities of mutant tRNAs and T-arms are used to analyze substrate recognition by pseudouridine 55 synthase. The 17-mer T-arm is an excellent substrate for the synthase, while disruption of the stem structure of the 17-mer T-arm eliminates activity. Kinetic data on tRNA mutants lacking single T-stem base pairs indicate that only the 53:61 base pair, which maintains the 7 base loop size, is essential for activity. The identities of individual bases in the stem are unimportant provided base pairing is intact. A major function of the T-stem appears to be the maintainence of a stable stem-loop structure and proper presentation of the T-loop to pseudouridine 55 synthase. The 7 base T-loop can be expanded or contracted by 1 base and still retains activity, albeit with a 30fold reduction in kcat. Kinetic analysis of T-loop mutants reveals the requirement for uridine54, uridine55, and adenine58, and a preference for cytosine over uridine at position 56
-
-
?
additional information
?
-
RNA containing 5-fluorouridine is a substrate
-
-
?
additional information
?
-
-
RNA containing 5-fluorouridine is a substrate
-
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
TruB is not inhibited by RNA containing 5-fluorouridine
-
additional information
-
TruB is not inhibited by RNA containing 5-fluorouridine
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
Km-values are determined for wild-type and mutant forms of yeast tRNAPhe
-
0.000124
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C58A/C174A/C193A
-
0.000127
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C174A
-
0.000146
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe. wild-type enzyme
-
0.000148
tRNA uridine55
pH 7.5, 37°C, Escherichia coli tRNAPhe, wild-type enzyme
-
0.000177
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C58A
-
0.000218
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C193V
-
0.000324
tRNA uridine55
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20G
-
0.000517
tRNA uridine55
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20L
-
0.0009
tRNA uridine55
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19R
-
0.00161
tRNA uridine55
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19M
-
0.0024
tRNA uridine55
pseudouridine synthase TruB with an 10-amino acid N-terminal truncation
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
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
-
0.011
tRNA uridine55
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19R
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0.012
tRNA uridine55
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19M
-
0.11
tRNA uridine55
pH 7.5, 37°C, Escherichia coli tRNAPhe, wild-type enzyme
-
0.11
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C58A/C174A/C193A
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0.12
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe. wild-type enzyme
-
0.16
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C174A
-
0.18
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C193V
-
0.23
tRNA uridine55
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20G
-
0.26
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C58A
-
0.52
tRNA uridine55
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20L
-
0.78
tRNA uridine55
pseudouridine synthase TruB with an 10-amino acid N-terminal truncation
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
7.43
tRNA uridine32
pH 7.5, 37°C, Escherichia coli tRNAPhe, wild-type enzyme
-
7.45
tRNA uridine32
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19M
-
12.2
tRNA uridine32
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme K19R
-
709.8
tRNA uridine32
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20G
-
1006
tRNA uridine32
pH 7.5, 37°C, Escherichia coli tRNAPhe, mutant enzyme P20L
-
821.9
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe. wild-type enzyme
-
825
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C193V
-
887
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C58A/C174A/C193A
-
1260
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C174A
-
1469
tRNA uridine55
pH 7.5, 37°C, substrate: Escherichia coli tRNAPhe, mutant enzyme C58A
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
physiological function
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
malfunction
-
a parallel lack of 2'-O-methylguanosine18 and pseudouridine55 in tRNA of Escherichia coli affects growth rate, translation of certain codons, sensitivity to amino acid analogs, and oxidation of some carbon compounds
malfunction
a truB null mutant grows normally on all growth media tested, but exhibits a competitive disadvantage in extended co-culture with its wild-type progenitor. The mutant phenotype can be complemented by both the cloned truB gene and by a D48C, catalytically inactive allele of truB. The truB mutant also exhibits a defect in survival of rapid transfer from 37 to 50°C
malfunction
deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
atomic-resolution crystal structure, TruB bound to RNA at 1.85 A resolution
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 tTruBRNA cocrystals. The resulting crystals are crushed and used as seeds for six subsequent sequential rounds of seeding into tTruB(D48N)RNA complex crystallization experiments
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C174A
kcat is 1.3fold higher than wild-type value, KM is 1.1fold lower than wild-type value
C193V
kcat is 1.5fold higher than wild-type value, KM is 1.5fold higher than wild-type value
C58A
kcat is 2.2fold higher than wild-type value, KM is 1.2fold higher than wild-type value
C58A/C174A/C193A
kcat is 1.1fold lower than wild-type value, KM is 1.2fold lower than wild-type value
K19M
kcat is 8fold lower than wild-type value. KM-value is 11fold higher than wild-type value
K19R
kcat is 10fold lower than wild-type value. KM-value is 6fold higher than wild-type value
P20G
kcat is 4.8fold lower than wild-type value. KM-value is 2.2fold higher than wild-type value
P20L
kcat is 2.1fold lower than wild-type value. KM-value is 3.5fold higher than wild-type value
D90A
-
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
-
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
-
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)
R181A
-
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
-
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
-
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)
additional information
additional information
mutant enzymes with substitution of the nearly invariant lysine and proline residues in motif I of RluA display only very mild kinetic impairment. Substitution of the aligned lysine and proline residues reduces structural stability
additional information
substitution of cysteine for amino acids with nonnucleophilic side chains does not significantly alter the catalytic activity
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ramamurthy, V.; Swann, S.; Spedaliere, C.; Mueller, E.
Role of cysteine residues in pseudouridine synhases of different families
Biochemistry
38
13106-13111
1999
Escherichia coli (P60340)
Hoang, C.; Ferre-D'Amare, A.R.
Cocrystal structure of a tRNA psi55 pseudouridine synthase: nucleotide flipping by an RNA-modifying enzyme
Cell
107
929-939
2001
Escherichia coli (P60340)
Pan, H.; Agarwalla, S.; Moustakas, D.T.; Finer-Moore, J.; Stroud, R.M.
Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit
Proc. Natl. Acad. Sci. USA
100
12648-12653
2003
Escherichia coli (P60340), Thermotoga maritima (Q9WZW0)
Hoang, C.; Hamilton, C.S.; Mueller, E.G.; Ferre-D'Amare, A.R.
Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA-sequestering peripheral protein domain
Protein Sci.
14
2201-2206
2005
Escherichia coli (P60340)
Gu, X.; Yu, M.; Ivanetich, K.M.; Santi, D.V.
Molecular recognition of tRNA by tRNA pseudouridine 55 synthase
Biochemistry
37
339-343
1998
Escherichia coli
Spedaliere, C.J.; Hamilton, C.S.; Mueller, E.G.
Functional importance of motif I of pseudouridine synthases: mutagenesis of aligned lysine and proline residues
Biochemistry
39
9459-9465
2000
Escherichia coli (P60340)
Urbonavicius, J.; Durand, J.M.; Bjrk, G.R.
Three modifications in the D and T arms of tRNA influence translation in Escherichia coli and expression of virulence genes in Shigella flexneri
J. Bacteriol.
184
5348-5357
2002
Escherichia coli, Shigella flexneri
Kinghorn, S.M.; O'Byrne, C.P.; Booth, I.R.; Stansfield, I.
Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance
Microbiology
148
3511-3520
2002
Escherichia coli (P60340)
Spedaliere, C.J.; Mueller, E.G.
Not all pseudouridine synthases are potently inhibited by RNA containing 5-fluorouridine
RNA
10
192-199
2004
Escherichia coli (P60340), Escherichia coli
Nurse, K.; Wrzesinski, J.; Bakin, A.; Lane, B.G.; Ofengand, J.
Purification, cloning, and properties of the tRNA PSI 55 synthase from Escherichia coli
RNA
1
102-112
1995
Escherichia coli (P60340), Escherichia coli
Gutgsell, N.; Englund, N.; Niu, L.; Kaya, Y.; Lane, B.G.; Ofengand, J.
Deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells
RNA
6
1870-1881
2000
Escherichia coli (P60340)
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