Information on EC 3.6.1.62 - 5'-(N7-methylguanosine 5'-triphospho)-[mRNA] hydrolase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.6.1.62
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RECOMMENDED NAME
GeneOntology No.
5'-(N7-methylguanosine 5'-triphospho)-[mRNA] hydrolase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
a 5'-(N7-methylguanosine 5'-triphospho)-[mRNA] + H2O = N7-methylguanosine 5'-diphosphate + a 5'-phospho-[mRNA]
show the reaction diagram
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SYSTEMATIC NAME
IUBMB Comments
5'-(N7-methylguanosine 5'-triphospho)-[mRNA] N7-methylguanosine-5'-diphosphate phosphohydrolase
Decapping of mRNA is a critical step in eukaryotic mRNA turnover. The enzyme is unable to cleave a free cap structure (m7GpppG) [3]. The enzyme from Vaccinia virus is synergistically activated in the presence of Mg2+ and Mn2+ [5].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
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
5'-(N7-methylguanosine 5'-triphospho)-mRNA + H2O
m7GDP + 5'-phospho-mRNA
show the reaction diagram
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luciferase mRNA
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?
m7G5'ppp5'-mRNA + H2O
m7GDP + 5'-phospho-mRNA
show the reaction diagram
m7G5'ppp5'-NO38 mRNA + H2O
m7GDP + 5'-phospho-NO38 mRNA
show the reaction diagram
removes m7G and m227G caps from RNAs, rendering them substrates for 5'-3' exonucleases for degradation in vivo. The metal identity determines both the efficiency of decapping and the RNA substrate specificity. In Mg2+ the protein hydrolyzes the 5' cap from only one RNA substrate: U8 small nucleolar RNA. In the presence of Mn2+ or Co2+ all RNAs are substrates and the decapping efficiency is higher. The metal that binds the X29/H29K proteins in vivo may determine whether these decapping proteins function solely as a negative regulator of ribosome biogenesis or can decap a wider variety of nuclear-limited RNAs
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?
m7G5'ppp5'-U3 snoRNA + H2O
m7GDP + 5'-phospho-U3 snoRNA
show the reaction diagram
removes m7G and m227G caps from RNAs, rendering them substrates for 5'-3' exonucleases for degradation in vivo. The metal identity determines both the efficiency of decapping and the RNA substrate specificity. In Mg2+ the protein hydrolyzes the 5' cap from only one RNA substrate: U8 small nucleolar RNA. In the presence of Mn2+ or Co2+ all RNAs are substrates and the decapping efficiency is higher. The metal that binds the X29/H29K proteins in vivo may determine whether these decapping proteins function solely as a negative regulator of ribosome biogenesis or can decap a wider variety of nuclear-limited RNAs
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?
m7G5'ppp5'-U3 snoRNA + H2O
m7GDP + U3 snoRNA + H2O
show the reaction diagram
removes m7G and m227G caps from RNAs, rendering them substrates for 5'-3' exonucleases for degradation in vivo. The metal identity determines both the efficiency of decapping and the RNA substrate specificity. In Mg2+ the protein hydrolyzes the 5' cap from only one RNA substrate: U8 small nucleolar RNA. In the presence of Mn2+ or Co2+ all RNAs are substrates and the decapping efficiency is higher. The metal that binds the X29/H29K proteins in vivo may determine whether these decapping proteins function solely as a negative regulator of ribosome biogenesis or can decap a wider variety of nuclear-limited RNAs
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?
m7G5'ppp5'-U8 snomRNA + H2O
m7GDP + 5'-phospho-U8 snomRNA
show the reaction diagram
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hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m7GDP and m227GDP from RNAs. hNUDT16 without the coordination of metals can not catalyze the hydrolytic reaction since no detectable cleaved products can be observed for either the U8 snoRNA or the mRNA substrate. Both Mg2+ and Mn2+ can effectively switch the protein from apoenzyme to holoenzyme. Mn2+ is more efficient as the activating factor
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?
m7G5'ppp5'-U8 snoRNA + H2O
m7GDP + 5'-phospho-U8 snoRNA
show the reaction diagram
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
m7G5'ppp5'-mRNA + H2O
m7GDP + 5'-phospho-mRNA
show the reaction diagram
m7G5'ppp5'-U8 snoRNA + H2O
m7GDP + 5'-phospho-U8 snoRNA
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zn2+
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confer only limited hydrolytic capability on hNUDT16. After 30 min incubation of the RNA substrate with the enzyme, only tiny amounts of m7GDP product are observed on the migration profiles
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Co2+
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supports D10p catalytic activity, but fails to demonstrate a synergistic effect on the enzyme when tested with Mn2+ ions
m7G5'ppp5'G
m7GTP
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inhibits decapping at large molar ratios relative to capped RNA substrate
Mg2+
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presence of two metal-ion-binding sites on the enzyme. Synergistic activation of the enzyme in the presence of Mg2+ and Mn2+ ions, suggesting the existence of two metal-ion binding sites on the D10 protein. One metal ion is co-ordinated by Glu132, while the second metal ion is co-ordinated by Glu145
Mn2+
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presence of two metal-ion-binding sites on the enzyme. Synergistic activation of the enzyme in the presence of Mg2+ and Mn2+ ions, suggesting the existence of two metal-ion binding sites on the D10 protein. One metal ion is co-ordinated by Glu132, while the second metal ion is co-ordinated by Glu145
N7-methylguanosine 5'-diphosphate
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D9 differs from D10 in having lower sensitivity to inhibition by nucleotide cap analogs unattached to RNA
N7-methylguanosine 5'-triphosphate
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D9 differs from D10 in having lower sensitivity to inhibition by nucleotide cap analogs unattached to RNA
poly(A) tail
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presence of a poly(A) tail reduces the level of decapping by more than 2fold. The inhibition of decapping is reversed upon the addition of poly(A) competitor
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uncapped RNA
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
DCP1 protein
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DCP2 interacts in vitro and in vivo with DCP1 and VARICOSE (VCS). The interacting proteins stimulate DCP2 activity, suggesting that the three proteins operate as a decapping complex. DCP1, DCP2, and VCS colocalize in cytoplasmic foci
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VARICOSE protein
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DCP2 interacts in vitro and in vivo with DCP1 and VARICOSE (VCS). The interacting proteins stimulate DCP2 activity, suggesting that the three proteins operate as a decapping complex. DCP1, DCP2, and VCS colocalize in cytoplasmic foci
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additional information
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hDcp2 requires the presence of an mRNA body attached to the cap to be active
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0034
m7G5'ppp5'-mRNA
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pH 7.5, 37°C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.063
m7G5'ppp5'G
Vaccinia virus
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pH 7.5, 37°C
0.3
m7GDP
Vaccinia virus
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pH 7.5, 37°C
0.003
m7GTP
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
embryonic fibroblast
Manually annotated by BRENDA team
; Dcp2 is present in embryonic brain, heart, liver, and kidney. A substantial decrease of Dcp2 is evident in heart, liver, and kidney at birth and a continual decrease to undetectable levels in the adult
Manually annotated by BRENDA team
; Dcp2 is present in embryonic brain, heart, liver, and kidney. A substantial decrease of Dcp2 is evident in heart, liver, and kidney at birth and a continual decrease to undetectable levels in the adult
Manually annotated by BRENDA team
; Dcp2 is present in embryonic brain, heart, liver, and kidney. A substantial decrease of Dcp2 is evident in heart, liver, and kidney at birth and a continual decrease to undetectable levels in the adult
Manually annotated by BRENDA team
additional information
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no activity in liver
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 22000, SDS-PAGE
dimer
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2 * 30000, SDS-PAGE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour-diffusion method
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strucutures of wild-type, and E198Q and E153Q catalytic glutamate mutants of the Dcp2 Nudix domain, to 2.1A, 1.8 A and 1.7 A resolution, respectively. Conserved glutamate residues E152, E153, and E198 coordinate a magnesium ion through a water mediated contact, while E149 directly contacts the metal. A conserved metal binding loop on the catalytic domain undergoes conformational changes during the catalytic cycle
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structure of the yeast enhancer of mRNA-decapping protein Edc3 LSm domain in complex with a short helical leucine-rich motif from subunit Dcp2. The motif interacts with the monomeric Edc3 LSm domain and recognizes a noncanonical binding surface. Additional helical leucine-rich motifs in the disordered C-terminal extension of Dcp2 can interact with Edc3. Both Edc3 and Scd6 stimulate decapping in vitro, presumably by preventing the Dcp1:Dcp2 complex from adopting an inactive conformation. The C-terminal helical leucine-rich motifs in Dcp2 are necessary for the localization of the Dcp1:Dcp2 decapping complex to P-bodies in vivo
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crystallizes as a homodimeric apoenzyme. Structure of X29 complexes with m7GpppA and pppG in the presence of Mn+2
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant D10 fusion protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(MBP)-D10 fusion is synthesized in Escherichia coli
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cloned into a His-tagged expression vector, overexpressed in Escherichia coli
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expresiion in Escherichia coli
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expression in Escherichia coli
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expression of a fragment of the yeast Dcp2 protein in Escherichia coli. This fragment contains the MutT/Nudix domain but lacks the non-conserved C-terminal tail and is also active in decapping
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overexpressed in Escherichia coli
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
Dcp2 expression is induced upon viral infection
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E148Q
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mutant enzyme is as stable as the wild-type enzyme
E76Q
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mutation totally abolishes the decapping activity under the standard assaying condition
E79Q
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mutation decreases the decapping activity of the enzyme
E80Q
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mutation totally abolishes the decapping activity under the standard assaying condition
R75L
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the mutant displays a weaker capability of hydrolysis
E153Q
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mutant has 3 molecules in the asymmetric unit. There is clear electron density for an octahedrally coordinated Mg2+ in the structure, similar to wild-type. Mutant is severely catalytically compromised and displays a linear dependence on pH over the range studied (pH 7–9.5)
E198Q
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mutant lacks clear density for a metal ion in the active site and fails to crystallize in the presence of any divalent cation
W50A
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Mutation in subunit Dcp2. While the wild-type Dcp1-Dcp2 complex is stimulated by enhancer of decapping Edc1CTR by a factor of 13, it only enhances catalysis in the W50A mutant by a factor of three. Coactivation of decapping by Dcp2 is linked to formation of the composite active site
E132A
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7.8% of wild-type activity. The E132A mutant shows a 5fold reduced affinity for Mg2+, but retains a Mn2+ affinity similar to that of the wild type
E141A
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10% of wild-type activity. The E141A mutant demonstrates only slight variations in both Mg2+ and Mn2+ binding
E141Q
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inactive mutant enzyme
E144Q/E145Q
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inactive mutant enzyme
E145A
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12.2% of wild-type activity. the E145A mutant shows 2-fold reduced affinity for Mg2+ and 6-fold reduced Mn2+ binding
E150Q
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mutant protein is inactive under all conditions
E89Q
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mutant protein is inactive under all conditions
E92Q
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mutant displays weak decapping activity under the standard decapping conditions in both Mg2+ and Mn2+, a significant increase in hydrolysis in the presence of higher concentrations of metal
E92Q/E93Q
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mutation displays less than 5% decapping activity in Mn2+ under the optimized decapping conditions. A 10fold increase in the amount of metal present in the reaction (to 1 mM Mn2+) only marginally increases the efficiency of cap hydrolysis
E93Q
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mutation displays less than 5% decapping activity in Mn2+ under the optimized decapping conditions. A 10fold increase in the amount of metal present in the reaction (to 1 mM Mn2+) only marginally increases the efficiency of cap hydrolysis
additional information
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decapping actiovity is abolished by point mutations in the Nudix hydrolase motif