Information on EC 6.5.1.4 - RNA 3'-terminal-phosphate cyclase (ATP)

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

EC NUMBER
COMMENTARY hide
6.5.1.4
-
RECOMMENDED NAME
GeneOntology No.
RNA 3'-terminal-phosphate cyclase (ATP)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + [RNA 3'-phosphate cyclase]-L-histidine = [RNA 3'-phosphate cyclase]-Ntau-(5'-adenylyl)-L-histidine + diphosphate
show the reaction diagram
(1a)
-
-
-
ATP + [RNA]-3'-(3'-phospho-ribonucleoside) = AMP + diphosphate + [RNA]-3'-(2',3'-cyclophospho)-ribonucleoside
show the reaction diagram
[RNA 3'-phosphate cyclase]-Ntau-(5'-adenylyl)-L-histidine + [RNA]-3'-(3'-phospho-ribonucleoside) = [RNA 3'-phosphate cyclase]-L-histidine + [RNA]-3'-ribonucleoside-3'-(5'-diphosphoadenosine)
show the reaction diagram
(1b)
-
-
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[RNA]-3'-ribonucleoside-3'-(5'-diphosphoguanosine) = [RNA]-3'-(2',3'-cyclophospho)-ribonucleoside + GMP
show the reaction diagram
(1c)
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-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cyclization
-
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esterification
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-
cyclic phosphomonoester
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Ligation
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-
-
-
SYSTEMATIC NAME
IUBMB Comments
RNA-3'-phosphate:RNA ligase (cyclizing, AMP-forming)
The enzyme converts the 3'-terminal phosphate of various RNA substrates into the 2',3'-cyclic phosphodiester in an ATP-dependent reaction. Catalysis occurs by a three-step mechanism, starting with the activation of the enzyme by ATP, forming a phosphoramide bond between adenylate and a histidine residue [5,6]. The adenylate group is then transferred to the 3'-phosphate terminus of the substrate, forming the capped structure [RNA]-3'-(5'-diphosphoadenosine). Finally, the enzyme catalyses an attack of the vicinal O-2' on the 3'-phosphorus, which results in formation of cyclic phosphate and release of the adenylate. The enzyme also has a polynucleotide 5' adenylylation activity [7]. cf. EC 6.5.1.5, RNA 3'-terminal-phosphate cyclase (GTP).
CAS REGISTRY NUMBER
COMMENTARY hide
85638-41-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
zebra fish
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-
Manually annotated by BRENDA team
fruit fly
-
-
Manually annotated by BRENDA team
mouse
-
-
Manually annotated by BRENDA team
no activity in Bacillus subtilis
-
-
-
Manually annotated by BRENDA team
no activity in Haemophilus influenzae
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-
-
Manually annotated by BRENDA team
no activity in Helicobacter pylori
-
-
-
Manually annotated by BRENDA team
no activity in Mycoplasma genitalium
-
-
-
Manually annotated by BRENDA team
no activity in Synechocystis sp.
-
-
-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
fission yeast
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + (Ap)npAp
AMP + ?
show the reaction diagram
-
pure oligoribonucleotide
-
?
ATP + (Up)10pGp
AMP + ?
show the reaction diagram
-
pure oligoribonucleotide
-
?
ATP + 5S rRNA
AMP + ?
show the reaction diagram
ATP + 5S rRNA-pCp
AMP + ?
show the reaction diagram
-
-
-
?
ATP + AAAACAAAAGp
AMP + ?
show the reaction diagram
-
-
?
ATP + AAAAUAAAAGCp
AMP + ?
show the reaction diagram
ATP + AAAAUAAAAGp
AMP + ?
show the reaction diagram
ATP + AAAAUAAAAGpCp
AMP + diphosphate + AAAAUAAAAGpC-2',3'-cyclic phosphate
show the reaction diagram
ATP + AUGp
AMP + ?
show the reaction diagram
-
pure oligoribonucleotide
-
?
ATP + CCCCACCCCGp
AMP + ?
show the reaction diagram
ATP + CCCCCACCCCGCp
AMP + ?
show the reaction diagram
-
pure oligoribonucleotide
-
?
ATP + DNA 5'-terminal-phosphate
AMP + AppDNA
show the reaction diagram
-
RtcA also catalyzes adenylylation of 5'-phosphate ends of DNA strands to form AppDNA product
-
-
?
ATP + human U14 snoRNA
AMP + ?
show the reaction diagram
ATP + oligodeoxyribonucleotide 3'-terminal phosphate
AMP + diphosphate + oligodeoxyribonucleotide-2',3'-cyclic-phosphate
show the reaction diagram
500fold poorer substrate than oligoribonucleotide 3'-terminal phosphate
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-
-
ATP + RNA 2'-terminal-phosphate
?
show the reaction diagram
ATP + RNA 3'-terminal-phosphate
?
show the reaction diagram
ATP + RNA 3'-terminal-phosphate
AMP + diphosphate + RNA terminal-2',3'-cyclic phosphate
show the reaction diagram
-
RtcA catalyzes the synthesis of RNA 2',3'-cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of RtcA with ATP to form a covalent RtcA-(histidinyl-N)-AMP intermediate and release diphosphate, transfer of AMP from RtcA to an RNA 3'-phosphate to form an RNA(3')pp(5')A intermediate, and attack by the terminal nucleoside O2' on the 3'-phosphate to form an RNA 2',3'-cyclic phosphate product and release AMP
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-
?
ATP + RNA 3'-terminal-phosphate
AMP + diphosphate + RNA terminal-2',3'-cyclic-phosphate
show the reaction diagram
ATP + RNA 3'-terminal-phosphate
AMP + diphosphate + RNA terminal-2',3'-cyclic-phosphodiester
show the reaction diagram
ATP + RNA 5'-terminal-phosphate
AMP + AppRNA
show the reaction diagram
-
RtcA also catalyzes adenylylation of 5'-phosphate ends of RNA strands to form AppRNA product
-
-
?
ATP + tobacco mosaic virus RNA
AMP + ?
show the reaction diagram
-
fragments modified by ligation of pGp, pAp, or pCp
-
?
ATP + tRNA-pNp
AMP + ?
show the reaction diagram
-
-
-
?
ATPgammaS + RNA 3'-terminal-phosphate
AMP + thiodiphosphate + RNA terminal-2',3'-cyclic-phosphate
show the reaction diagram
CTP + RNA 3'-terminal-phosphate
CMP + diphosphate + RNA terminal-2',3'-cyclic-phosphate
show the reaction diagram
GTP + RNA 3'-terminal-phosphate
GMP + diphosphate + RNA terminal-2',3'-cyclic-phosphate
show the reaction diagram
RNA-N3'p + ATP
RNA-N-2',3'-cyclic phosphate + AMP + diphosphate
show the reaction diagram
-
-
-
-
ir
UTP + RNA 3'-terminal-phosphate
UMP + diphosphate + RNA terminal-2',3'-cyclic-phosphate
show the reaction diagram
additional information
?
-
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
ATP + RNA 3'-terminal-phosphate
?
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
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only 5% of the activation by Mg2+
K+
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optimal concentration: 150-200 mM
Na+
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optimal concentration: 150-200 mM
additional information
-
no activity with Ca2+, Zn2+, or Cu2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
iodoacetamide
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-
SDS
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Sodium phosphate
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Vanadyl-ribonucleoside complex
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0017 - 0.02
ATP
0.006
ATPgammaS
0.1 - 0.2
GTP
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
?
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the rate of 2'-phosphate cyclization by RtcA is five orders of magnitude slower than 3'-phosphate cyclization, notwithstanding that RtcA binds with similar affinity to RNA3'p and RNA2'p substrates
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.021
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-
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 6
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pH-optimum for DNA 5'-adenylylation Tris acetate buffer
8 - 8.5
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pH-optimum for RNA 3'-cyclization
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8 - 10.5
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8.0-9.0: maximal activity, 10.5: 70% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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similar rates of reaction at 25°C, 30°C, and 37°C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 37
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0°C: 25% of maximal activity, 12°C: 60% of maximal activity, 25-37°C: maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
Escherichia coli (strain K12);
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3);
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38000 - 40000
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glycerol density gradient sedimentation, gel filtration
39000
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SDS-PAGE
39400
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calculated from cDNA
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
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the apoenzyme of Escherichia coli RtcA crystallizes as a disulfide-linked homodimer and reveales a fold composed of four tandem modules, each comprising a four-stranded beta sheet overlying two alpha helices, X-ray diffraction
monomer
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1 * 38000-40000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion technique, 2 orthorhombic crystal forms, space group P2(1)2(1)2(1), unit-cell dimensions a : 101.8 A, b : 126.6 A and c : 128.8 A, and P2(1)2(1)2 with unit cell dimensions a : 125.8 A, b : 133.5 A, c : 51.0 A
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crystal structure of the enzyme in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket
crystallized in the states St-Rtc, St-Rtc+Mn, St-Rtc+ATP, St-Rtc+AMP and St-Rtc-AMP
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native enzyme, with AMP covalently bound, in complex with AMP, with ATP, or Mn2+, at 2.25 A, 2.25A, 2.9 A, 2.4 A and 3.2 A resolutions, respectively. Upon binding of Mg2+ to residue E10, the triphosphate group of the trapped ATP changes its conformation, with help of ligands R17 and R39. The Nepsilon atom of H307 attacks the alpha-phosphate group to form a new P-N bond. When a truncated RNA is bound, its 3'-phosphate group may be forced to react with the phosphate group of AMP, and the activiated 3'-phosphate group may be attacked by the 2'-hydroxyl group to generate the 2',3'-cyclic phosphodiester
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
3 d, 50% loss of activity
20
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3-4 h, 50% loss of activity
42
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5 min, 55% loss of activity
50
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5 min, 95% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
not affected by several cycles of freezing and thawing
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resistant to protease digestion
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70°C, stable for at least 3 months
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-70°C, stable for at least 6 months
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4°C, 50% loss of activity after 3 days
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme
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using Ni-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
bacterially overexpressed as a fusion protein
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cDNA characterized
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cDNA characterized, complements yeast strains depleted of Rcl1p
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cloned and overexpressed
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cloned and overexpressed in Escherichia coli as fusion protein with glutathione S-transferase
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expressed in Escherichia coli
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expressed in Escherichia coli as a His-tagged fusion protein
expressed in Escherichia coli BL21-Codon Plus (DE3)
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expressed sequence tags encoding cyclase like protein identified
expression in Escherichia coli
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gene encoding cyclase like protein present
gene rtcA cloned and overexpressed
gene rtcA encoding cyclase cloned and overexpressed
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overexpressed in Escherichia coli
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overexpression in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C308A
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recombinant mutant protein
D287A
-
activity very low, 1% of wild-type enzyme
E270A
-
reduced activity compared to wild-type enzyme, about 80%
F135A
-
activity very low, 3% of wild-type enzyme
F251A
-
considerably reduced activity compared to wild-type enzyme, about 30%
H309G
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enzyme inactive
P131G
-
considerably reduced activity compared to wild-type enzyme, about 35%
Q104A
-
almost same activiy like wild-type enzyme
Q288A
-
activity very low, 2% of wild-type enzyme
S129A
-
almost same activiy like wild-type enzyme
Y284A
-
considerably reduced activity compared to wild-type enzyme (12%)
D297A
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enzyme inactive
E14A
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activity very low, only 4% of RNA is cyclized compared to 95% with wild-type enzyme
H326A
-
almost same activiy like wild-type enzyme
H52A
-
half activity compared to wild-type enzyme
Q51A
-
almost same activiy like wild-type enzyme
R21A
-
enzyme inactive
R40A
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enzyme inactive
R43A
-
enzyme inactive
Y294A
-
enzyme inactive
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