Information on EC 6.5.1.8 - 3'-phosphate/5'-hydroxy nucleic acid ligase

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

EC NUMBER
COMMENTARY hide
6.5.1.8
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RECOMMENDED NAME
GeneOntology No.
3'-phosphate/5'-hydroxy nucleic acid ligase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(ribonucleotide)n-2',3'-cyclophosphate + 5'-hydroxy-(ribonucleotide)m + GTP + H2O = (ribonucleotide)n+m + GMP + diphosphate
show the reaction diagram
(2) overall reaction
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(ribonucleotide)n-2',3'-cyclophosphate + H2O = (ribonucleotide)n-3'-phosphate
show the reaction diagram
(2a)
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(ribonucleotide)n-3'-(5'-diphosphoguanosine) + 5'-hydroxy-(ribonucleotide)m = (ribonucleotide)n+m + GMP
show the reaction diagram
(1c); (2d)
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(ribonucleotide)n-3'-phosphate + 5'-hydroxy-(ribonucleotide)m + GTP = (ribonucleotide)n+m + GMP + diphosphate
show the reaction diagram
(1) overall reaction
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5'-guanosyl [RNA ligase]-Ntau-phosphono-L-histidine + (ribonucleotide)n-3'-phosphate = (ribonucleotide)n-3'-(5'-diphosphoguanosine) + [RNA ligase]-L-histidine
show the reaction diagram
(1b); catalytic reaction mechanism, detailed overview
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GTP + [RNA ligase]-L-histidine = 5'-guanosyl [RNA ligase]-Ntau-phosphono-L-histidine + diphosphate
show the reaction diagram
(1a); (2b)
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
tRNA splicing II
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SYSTEMATIC NAME
IUBMB Comments
poly(ribonucleotide)-3'-phosphate:5'-hydroxy-poly(ribonucleotide) ligase (GMP-forming)
The enzyme is a GTP- and Mn2+-dependent 3'-5' nucleic acid ligase with the ability to join RNA with 3'-phosphate or 2',3'-cyclic-phosphate ends to RNA with 5'-hydroxy ends. It can also join DNA with 3'-phosphate ends to DNA with 5'-hydroxy ends, provided the DNA termini are unpaired [6]. The enzyme is found in members of all three kingdoms of life, and is essential in metazoa for the splicing of intron-containing tRNAs. The reaction follows a three-step mechanism with initial activation of the enzyme by GTP hydrolysis, forming a phosphoramide bond between the guanylate and a histidine residue. The guanylate group is transferred to the 3'-phosphate terminus of the substrate, forming the capped structure [DNA/RNA]-3'-(5'-diphosphoguanosine). When a suitable 5'-OH end is available, the enzyme catalyses an attack of the 5'-OH on the capped end to form a 3'-5' phosphodiester splice junction, releasing the guanylate. When acting on an RNA 2',3'-cyclic-phosphate, the enzyme catalyses an additional reaction, hydrolysing the cyclic phosphate to a 3'-phosphate [9]. The metazoan enzyme requires activating cofactors in order to achieve multiple turnover catalysis [8].
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(ribonucleotide)10-3'-phosphate + 5'-hydroxy-(ribonucleotide)10 + dGTP
(ribonucleotide)20 + dGMP + diphosphate
show the reaction diagram
(ribonucleotide)10-3'-phosphate + 5'-hydroxy-(ribonucleotide)10 + GTP
(ribonucleotide)20 + GMP + diphosphate
show the reaction diagram
(ribonucleotide)10-3'-phosphate + 5'-hydroxy-(ribonucleotide)10 + ITP
(ribonucleotide)20 + IMP + diphosphate
show the reaction diagram
(ribonucleotide)20-3'-phosphate + 5'-hydroxy-(ribonucleotide)20 + GTP
(ribonucleotide)40 + GMP + diphosphate
show the reaction diagram
(ribonucleotide)n-2',3'-cyclophosphate + 5'-hydroxy-(ribonucleotide)m
(ribonucleotide)n+m
show the reaction diagram
(ribonucleotide)n-2',3'-cyclophosphate + 5'-hydroxy-(ribonucleotide)m + GTP + H2O
(ribonucleotide)n+m + GMP + diphosphate
show the reaction diagram
(ribonucleotide)n-2',3'-cyclophosphate + 5'-hydroxy-(ribonucleotide)m + GTP + H2O
(ribonucleotide)n+m + GMP + diphosphate |
show the reaction diagram
(ribonucleotide)n-2',3'-cyclophosphate + H2O
(ribonucleotide)n-3'-phosphate
show the reaction diagram
(ribonucleotide)n-3'-(5'-diphosphoguanosine) + 5'-hydroxy-(ribonucleotide)m
(ribonucleotide)n+m + GMP
show the reaction diagram
(ribonucleotide)n-3'-phosphate + 5'-hydroxy-(ribonucleotide)m + GTP
(ribonucleotide)n+m + GMP + diphosphate
show the reaction diagram
(ribonucleotide)n-3'-phosphate + 5'-phospho-(ribonucleotide)m + GTP
(ribonucleotide)n+m + GMP + diphosphate
show the reaction diagram
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overall reaction
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?
(ribonucleotide)n-3'-phosphate + GTP
? + GMP
show the reaction diagram
(truncated 16S rRNA)-3'-phosphate + 5'-hydroxy-RNA43 + GTP
16S rRNA + GMP + diphosphate
show the reaction diagram
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endoribonuclease MazF, the toxin component of the toxin-antitoxin module mazEF, specifically cleaves the 16S rRNA of 70S ribosomes at an ACA site located at positions 15001502. Thereby, the ribosome loses a 3'-terminal 16S rRNA fragment of 43 nucleotides. RNA ligase RtcB catalyzes the religation of the truncated 16S rRNA present in specialized ribosomes. Thereby their ability to translate canonical mRNAs is fully restored
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5'-guanosyl [RNA ligase]-NT-phosphono-L-histidine + (ribonucleotide)n-3'-phosphate
(ribonucleotide)n-3'-(5'-diphosphoguanosine) + [RNA ligase]-L-histidine
show the reaction diagram
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RtcB transfers GMP to the RNA 3'-phosphate end
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?
5'-guanosyl [RNA ligase]-Ntau-phosphono-L-histidine + (ribonucleotide)n-3'-phosphate
(ribonucleotide)n-3'-(5'-diphosphoguanosine) + [RNA ligase]-L-histidine
show the reaction diagram
GTP + [RNA ligase]-L-histidine
5'-guanosyl [RNA ligase]-NT-phosphono-L-histidine + diphosphate
show the reaction diagram
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?
GTP + [RNA ligase]-L-histidine
5'-guanosyl [RNA ligase]-Ntau-phosphono-L-histidine + diphosphate
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00018 - 0.00055
(ribonucleotide)10-3'-phosphate
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0.0014 - 0.0122
(ribonucleotide)20-3'-phosphate
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
a subset of RtcB is associated with the endoplasmic reticulum
Manually annotated by BRENDA team
a subset of RtcB is associated with the nuclear envelope
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45000
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gel fitlration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
three structures of RtcB complexes that capture snapshots along the entire guanylylation pathway. RtcB in complex with Mn(II) and GTP analogue guanosine 5'-(alpha-thio)-triphosphate shows that Mn1 is poised to stabilize the pentavalent transition state of guanylylation while a second manganese ion (Mn2) is coordinated to a nonbridging oxygen of the gamma-phosphoryl group. The diphosphate leaving group of 5'-(alpha-thio)-triphosphate is oriented apically to His404 with the epsilon nitrogen poised for in-line attack on the alpha phosphorus atom. The structure of RtcB in complex with 5'-(alpha-thio)-triphosphate also reveals the network of hydrogen bonds that recognize GTP and shows significant conformational changes accompanying the binding of the cofactor. A structure of the enzymic histidine-GMP intermediate depicts the end of the guanylylation pathway
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crystal structures of RtcB in complex with Mn2+ alone and together with a covalently bound GMP, to 1.6 A and 2.3 A resolution, respectively. The RtcB/Mn2+ structure shows two Mn2+ ions at the active site, and an array of sulfate ions nearby that indicate the binding sites of the RNA phosphate backbone. The structure of the RtcB-GMP/Mn2+ complex reveals the detailed geometry of guanylylation of histidine 404. The enzyme's substrate-induced GTP binding site and the putative reactive RNA ends are in the vicinity of the binuclear Mn2+ active center
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression in Saccharomyces cerevisiae
exprression in Escherichia coli
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exprression in Escherichia coli; exprression in Escherichia coli
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
RtcB mRNA is processed by endoribonuclease MazF and selectively translated during stress
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C78A
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almost complete loss of activity
C78S
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complete loss of activity
D75A
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almost complete loss of activity
D75E
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complete loss of activity
D75N
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complete loss of activity
H168A
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almost complete loss of activity
H168N
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complete loss of activity
H168Q
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complete loss of activity
H185A
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activity is severely impaired
H280A
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active, protein is able to complement a Saccharomyces cerevisiae Trl1 mutant
K298A
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activity is severely impaired
N167A
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activity is severely impaired
R189A
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activity is severely impaired; almost complete loss of activity
R341A
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almost complete loss of activity
C100A
inactive for ligation reaction
H205A
inactive for ligation reaction
H236A
inactive for ligation reaction
C100A
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inactive for ligation reaction
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H205A
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inactive for ligation reaction
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H236A
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inactive for ligation reaction
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C98A
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mutation in predicted metal-binding site, loss of activity