Information on EC 5.4.99.12 - tRNA pseudouridine38-40 synthase

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

EC NUMBER
COMMENTARY
5.4.99.12
-
RECOMMENDED NAME
GeneOntology No.
tRNA pseudouridine38-40 synthase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
tRNA uridine38-40 = tRNA pseudouridine38-40
show the reaction diagram
-
-
-
-
tRNA uridine38-40 = tRNA pseudouridine38-40
show the reaction diagram
the mechanism of pseudouridine synthase I is deduced from its interaction with 5-fluorouracil-tRNA. The covalent complex formed between pseudouridine synthase I and 5-fluorouracil-tRNA involves Michael adduct formation between Asp60 of pseudouridine synthase I and the 6-carbon of 5-fluorouracil39 of tRNA to form a covalent pseudouridine synthase I-5-fluorouracil-tRNA complex
-
tRNA uridine38-40 = tRNA pseudouridine38-40
show the reaction diagram
structural, computational, and functional studies provide the basis for a substrate recognition model for the regional selectivity. By binding to the conserved parts of tRNAs (elbow and D stem backbone), TruA recognizes multiple tRNAs independent of sequence variations. Anchored at these two regions, TruA positions the anticodon stem loop near the active site without constraining its flexibility, thereby increasing the effective concentration of each target position, 38, 39, and 40, in the vicinity of the active site. The thermal motions of the anticodon stem loop allow the nucleotides at each of the three sites to be dynamically accessible for modification. TruA utilizes the intrinsic flexibility of the anticodon stem loop for site promiscuity and also to select against intrinsically stable tRNAs
-
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
-
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].
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
hisT
-
gene name
mPus3p
Q9JI38
-
pseudouridine synthase
-
-
pseudouridine synthase 3
Q9JI38
-
pseudouridine synthase I
-
-
pseudouridine synthase I
P07649
-
pseudouridine synthase I
-
-
PUS3
P31115
gene name
tRNA pseudouridine synthase 3
P31115
-
tRNA pseudouridine synthase I
-
-
tRNA pseudouridine synthase I
P07649
-
TruA
-, P07649
-
CAS REGISTRY NUMBER
COMMENTARY
430429-15-5
-
61506-89-6
-
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
malfunction
-
disruption of the DEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature, 37 C
malfunction
-
deletion of the PUS3 gene has an effect on the efficiency of the translation process. Reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs
malfunction
-
hisT mutants of Salmonella typhimurium lack the enzyme that modifies uridine to pseudouridine in the anticodon regions of many tRNAs. The regulation of a large number of amino acid biosynthetic pathways is altered by the hisT mutation
malfunction
O87016
tRNA pseudouridine synthase is able to complement the type III gene expression defect of the fimV mutant. Thus fimV and truA form an operon and fimV mutation has a polar effect on truA. A truA mutant is defective in type III gene expression while its twitching motility is unaffected, and a truA clone is able to complement the type III secretion defect
physiological function
-
deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In the mutant, there is a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA. The presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast
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; 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
O87016
the truA gene of Pseudomonas aeruginosa is required for the expression of type III secretory genes
physiological function
-
Pus3p is unique in its ability to modulate frameshifting and readthrough events during translation. This aspect of its activity may be responsible for HOT1 recombination phenotypes observed in deg1 mutants
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Escherichia coli tRNAPhe uridine39
Escherichia coli tRNAPhe pseudouridine39
show the reaction diagram
-
-
-
-
?
human tRNALeu uridine 38 uridine39
human tRNALeu pseudouridine 38 pseudouridine39
show the reaction diagram
-, Q9JI38
-
psuedouridine formation in position 39 is clearly preferred over position 38
-
?
human tRNALys3
?
show the reaction diagram
-, Q9JI38
-
-
-
?
Salmonella typhimurium HisT- tRNALeu
?
show the reaction diagram
-
tRNAPhe2 from mutant HisT- strain defective for tRNA pseudouridine synthase I that forms pseudouridine in the 3'-side of the anticodon region of approximately half of the cellular tRNAs
-
-
?
Salmonella typhimurium HisT- tRNAPhe2 uridine39
Salmonella typhimurium HisT- tRNAPhe2 pseudouridine39
show the reaction diagram
-
tRNAPhe2 from mutant HisT- strain defective for tRNA pseudouridine synthase I that forms pseudouridine in the 3 side of the anticodon region of approximately half of the cellular tRNAs
specific modification of uridine39
-
?
Salmonelly typhimurium HisT- tRNATyr
?
show the reaction diagram
-
tRNAPhe2 from mutant HisT- strain defective for tRNA pseudouridine synthase I that forms pseudouridine in the 3'-side of the anticodon region of approximately half of the cellular tRNAs
-
-
?
tRNA uridine38
tRNA pseudouridine38
show the reaction diagram
P31115, -
-
-
-
?
tRNA uridine38
tRNA pseudouridine38
show the reaction diagram
-
-
-
-
?
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
-
-
?
tRNA uridine38
tRNA pseudouridine38
show the reaction diagram
-
the enzyme modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine
-
-
?
tRNA uridine38-40
tRNA pseudouridine38-40
show the reaction diagram
-
-
-
-
?
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
-
-
?
tRNA uridine38-40
tRNA pseudouridine38-40
show the reaction diagram
-
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
-
-
?
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. The molecular basis for the site and substrate promiscuity is studied by determining the crystal structures of Eschrichia coli TruA in complex with two different leucyl tRNAs in conjunction with functional assays and computer simulation
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
P31115, -
-
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
-
-
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
-
modified tRNALeu3 with uridine at position 39 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
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
-
tRNAPhe from yeast, which contains a single target for tRNA pseudouridine synthase I at U39
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
-
tRNAPhe with uridine at position 39
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
-
the enzyme modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
-, Q9JI38
with tRNA substrates from both yeast and humans, uridines at position 39 are modified to pseudouridine. In a tRNA substrate with a uridine at position 38 (human tRNALeu), there is very slight formation of pseudouridine at that position after incubation with mPus3p
-
-
?
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
-
-
?
tRNAHis guanidine36 uridine38 cytidine39
RNAHis guanidine36 pseudouridine38 cytidine39
show the reaction diagram
-
-
-
-
?
tRNAHis uridine38 cytidine39
RNAHis pseudouridine38 cytidine39
show the reaction diagram
-
-
-
-
?
tRNAHis uridine38 uridine39
tRNAHis pseudouridine38 pseudouridine39
show the reaction diagram
-
-
-
-
?
tRNALeu3
?
show the reaction diagram
-
-
-
-
?
tRNALeu3 carrying uridine at position 38
tRNALeu3 carrying pseudouridine at position 38
show the reaction diagram
-
-
-
-
?
tRNALeu3 carrying uridine at position 39
tRNALeu3 carrying pseudouridine at position 39
show the reaction diagram
-
-
-
-
?
tRNALeu3 carrying uridine at position 40
tRNALeu3 carrying pseudouridine at position 40
show the reaction diagram
-
-
-
-
?
tRNAVal2a cytidine36 uridine38
tRNAVal2a cytidine36 pseudouridine38
show the reaction diagram
-
poor substrate, tRNAVal2a is not a substrate
-
-
?
yeast tRNAPhe
?
show the reaction diagram
-, Q9JI38
-
-
-
?
human tRNASer uridine39
human tRNASer pseudouridine39
show the reaction diagram
-, Q9JI38
-
-
-
?
additional information
?
-
-
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
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
tRNA uridine38
tRNA pseudouridine38
show the reaction diagram
P31115, -
-
-
-
?
tRNA uridine38
tRNA pseudouridine38
show the reaction diagram
-
-
-
-
?
tRNA uridine38
tRNA pseudouridine38
show the reaction diagram
-
the enzyme modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine
-
-
?
tRNA uridine38-40
tRNA pseudouridine38-40
show the reaction diagram
-
-
-
-
?
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
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
P31115, -
-
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
-
-
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
-
the enzyme modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine
-
-
?
tRNA uridine39
tRNA pseudouridine39
show the reaction diagram
-, Q9JI38
with tRNA substrates from both yeast and humans, uridines at position 39 are modified to pseudouridine. In a tRNA substrate with a uridine at position 38 (human tRNALeu), there is very slight formation of pseudouridine at that position after incubation with mPus3p
-
-
?
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
additional information
-
no cofactors required
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
a monovalent cation is required for maximum activity
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
5-fluorouracil tRNAPhe
-
enzyme forms a covalent adduct with 5-fluorouracil-tRNA to form a putative analog of a steady-state intermediate in the normal reaction pathway. The putative Asp nucleophile is attached to the 6-position of the target 5-fluorouracil-tRNA to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage, and the intermediate contains an intact N-glycosidic bond linking the modified base to the polynucleotide chain
-
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
-
Calf thymus DNA
-
native and denatured, about 66% residual activity
-
Dithionitrobenzoate
-
irreversibly inactivates
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 15C prior to addition of substrate, there is a time-dependent inactivation of the enzyme with a half-life of 35 min
-
iodoacetate
-
irreversibly inactivates
p-chloromercuribenzoate
-
irreversibly inactivates
poly(rA)n
-
71% residual acivity
-
poly(rC)n
-
93% residual acivity
-
poly(rG)n
-
3.2% residual acivity
-
poly(rI)n
-
11.9% residual acivity
-
poly(rU)n
-
44% residual acivity
-
tRNA
-
activity is inhibited by single tRNAs or bulk tRNA from all sources tested, and by ribosomal RNA
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
additional information
-
a thiol is required for maximum activity
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.000086
-
Escherichia coli tRNAPhe uridine39
-
wild-type, pH 8.0, 30C
-
0.00011
-
Escherichia coli tRNAPhe uridine39
-
mutant C55A/C154A/C169A, pH 8.0, 30C
-
0.000086
-
tRNA uridine39
-
pH 8.0, 30, wild-type enzyme
-
0.00011
-
tRNA uridine39
-
pH 8.0, 30C, mutant enzyme C55A/C154A/C169A
-
0.00094
-
tRNA uridine39
-
pH 8.0, 22C
-
0.0018
-
tRNA uridine39
-
pH 8.0, 15C
-
0.0000053
-
tRNAHis guanidine36 uridine38 cytidine39
-
pH 8.0, 30C
-
0.0000804
-
tRNAHis uridine38 cytidine39
-
pH 8.0, 30C
-
0.0000456
-
tRNAHis uridine38 uridine39
-
pH 8.0, 30C
-
0.000669
-
tRNAVal2a cytidine36 uridine38
-
pH 8.0, 30C
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.18
-
tRNA uridine39
-
pH 8.0, 15C
-
0.73
-
tRNA uridine39
-
pH 8.0, 22C
-
0.0019
-
tRNAHis guanidine36 uridine38 cytidine39
-
pH 8.0, 30C
-
0.0746
-
tRNAHis uridine38 cytidine39
-
pH 8.0, 30C
-
0.144
-
tRNAHis uridine38 uridine39
-
pH 8.0, 30C
-
0.0244
-
tRNAVal2a cytidine36 uridine38
-
pH 8.0, 30C
-
kcat/KM VALUE [1/mMs-1]
kcat/KM VALUE [1/mMs-1] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
160
-
tRNA uridine38
-
pH 8.0, wild-type enzyme
0
100
-
tRNA uridine39
-
pH 8.0, 15C
0
200
-
tRNA uridine39
-
pH 8.0, wild-type enzyme
0
777
-
tRNA uridine39
-
pH 8.0, 22C
0
860
-
tRNA uridine40
-
pH 8.0, wild-type enzyme
0
0.16
-
tRNALeu3
-
pH 8.0, 22C
0
0.2
-
tRNALeu3 carrying uridine at position 39
-
pH 8.0, 22C
0
0.86
-
tRNALeu3 carrying uridine at position 40
-
pH 8.0, 22C
0
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0001
-
fluorouracil-substituted tRNA
-
-
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8
-
-
assay at
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
30
-
-
activity of yeast extract and purified yeast enzyme
30
-
-
assay at
37
-
-
activity of Escherichia coli extract
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-, Q9JI38
x * 55552, calculated
?
-
x * 31000, SDS-PAGE
dimer
-
; crystallization data
homodimer
-
homodimeric structure in crystals
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
; 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
-
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; 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
-
hanging-drop vapor diffusion method at 293 K. The crystals have a stick-like shape, and belong to the space group P4(1)2(1)2, with unit cell dimensions of a = b = 91.5 A and c = 164.0 A. Crystal structure is determined at 2.25 A resolution
-
to 2.25 A resolution. Structure reveals the remarkably flexible structural features in the tRNA-binding cleft, which may be responsible for the primary tRNA interaction. The charged residues occupying the intermediate positions in the cleft may lead the tRNA to the active site for catalysis. The tRNA probably makes the melting base pairs move into the cleft, and a conformational change of the substrate tRNA may be necessary to facilitate access to the active site aspartate residue, deep within the cleft
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
22
-
-
1 h, about 50% loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-20C or 2C, 50% w/v glycerol, 50% loss of activity after 2 years
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
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
-
wild-type and mutant enzymes D60A, D60S, D60E, D60N, and D60K
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
; pseudouridine synthase I is cloned behind a T7 promoter and expressed in Escherichia coli to about 20% of total soluble proteins
-
-
-, Q9JI38
expression in Escherichia coli
-
expression in Escherichia coli
-
EXPRESSION
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
hisT transcription is positively growth rate regulated in exponentially growing bacteria and is induced during the transition from exponential to stationary growth phase
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
C154A
-
activity similar to wild-type; mutant shows high levels of activity
C154S
-
complete loss of activity, mutant is capable of binding to substrate tRNAPhe; no activity
C169A
-
activity similar to wild-type; mutant shows high levels of activity
C169S
-
complete loss of activity, mutant is unable to bind substrate tRNAPhe; no activity
C55A/C154A/C169A
-
activity similar to wild-type; only a small change in both Km and Vmax parameters as compared with wild-type enzyme. These mutations cause a 1.2fold increase in Km and a 1.6fold decrease in Vmax. The overall Vmax/Km ratio is lowered by a factor of 2 for the triple mutant
C55S
-
activity similar to wild-type; maintains activity levels similar to those of wild-type enzyme
D60A
-
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA; mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60E
-
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA; mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60K
-
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA; mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
D60N
-
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA; mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
R58A
-
inactive; 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
D151A
-
inactive; inactive mutant
D60S
-
catalytically inactive, fails to form covalent complexes with fluorouracil-substituted tRNA; mutants binds tRNA but is catalytically inactive and fails to form covalent complexes with fluorouracil-substituted tRNA
additional information
-
all Asp60 mutants bind tRNA but are catalytically inactive and fail to form covalent complexes with fluorouracil-substituted tRNA. It is concluded that the conserved Asp60 is essential for pseudouridine synthase activity