Information on EC 3.1.13.5 - ribonuclease D

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

EC NUMBER
COMMENTARY
3.1.13.5
-
RECOMMENDED NAME
GeneOntology No.
ribonuclease D
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
exonucleolytic cleavage that removes extra residues from the 3'-terminus of tRNA to produce 5'-mononucleotides
show the reaction diagram
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
tRNA processing
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
RNase D
-
-
RNase D
Q38DE2
-
RNase D
Trypanosoma brucei 29-13
Q38DE2
-
-
CAS REGISTRY NUMBER
COMMENTARY
9073-62-5
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
strain K-12
-
-
Manually annotated by BRENDA team
-
Q38DE2
UniProt
Manually annotated by BRENDA team
Trypanosoma brucei 29-13
-
Q38DE2
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
evolution
Q38DE2
TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain
evolution
Trypanosoma brucei 29-13
-
TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain
-
malfunction
Q38DE2
TbRND depletion results in gRNA tails extended by 2-3 nucleotides on average. Second, overexpression of wild-type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3'-uridylated, are unaffected by TbRND depletion or overexpression, phenotypes, overview
malfunction
Trypanosoma brucei 29-13
-
TbRND depletion results in gRNA tails extended by 2-3 nucleotides on average. Second, overexpression of wild-type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3'-uridylated, are unaffected by TbRND depletion or overexpression, phenotypes, overview
-
physiological function
Q38DE2
TbRND functions in guide RNA metabolism in vivo, TbRND is a 3' to 5' exoribonuclease that functions highly specific to the mitochondrion of trypanosomes. In vitro, TbRND exhibits 3' to 5' exoribonuclease activity, with specificity toward uridine homopolymers, including the 3' oligo(U) tails of guide RNAs that provide the sequence information for RNA editing
physiological function
Trypanosoma brucei 29-13
-
TbRND functions in guide RNA metabolism in vivo, TbRND is a 3' to 5' exoribonuclease that functions highly specific to the mitochondrion of trypanosomes. In vitro, TbRND exhibits 3' to 5' exoribonuclease activity, with specificity toward uridine homopolymers, including the 3' oligo(U) tails of guide RNAs that provide the sequence information for RNA editing
-
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
structurally altered tRNA
5'-mononucleotide + ?
show the reaction diagram
-
exonuclease activity. RNase D can recognize structurally altered tRNA molecules. The enzyme acts poorly on intact tRNA and is inactive with the synthetic polyribonucleotides, poly(A), poly(U), or double-stranded poly(A)*poly(U). The enzyme acts on diesterase-treated tRNA, but relatively poorly on intact tRNA. RNase D does not attack ribosomal RNA
-
-
?
tRNA containing extra residues at the 3'-terminus
5'-mononucleotide + tRNA
show the reaction diagram
-
-
-
-
?
tRNA containing extra residues at the 3'-terminus
5'-mononucleotide + tRNA
show the reaction diagram
-
ribonuclease D is not essential for the normal growth of Escherichia coli or bacteriophage T4 or for the biosynthesis of a T4 suppressor tRNA
-
-
?
tRNA containing extra residues at the 3'-terminus
5'-mononucleotide + tRNA
show the reaction diagram
-
the enzyme can act at the 3'-terminus of tRNA in vivo. RNase D participates in tRNA metabolism
-
-
?
tRNA-C-C-A-Cn + H2O
tRNA-C-C-A-Cn-1 + cytosine
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
enzyme is involved in structured RNA processing
-
-
-
additional information
?
-
-
the enzyme may represent an intracellular scavenging mechanism for denatured tRNAs and other inactive RNA molecules
-
-
-
additional information
?
-
-
alteration of the 3'-terminal base has no effect on the rate of hydrolysis, whereas modification of the 3'-terminal sugar has a major effect. tRNA terminating with a 3'-phosphate is completely inactive as a substrate. The rate of hydrolysis of intact tRNA is very slow compared to tRNAs containing extra residues or compared to tRNAs from which part of the -C-C-A sequence has been removed. Oxidation of the terminal sugar, reduction of the dialdehyde with borohydride, or removal of the terminal AMP from intact tRNA increase the activity of the substrate. Addition of a second -C-C-A sequence gives an active substrate indicating that the relative resistance of intact tRNA to RNase D hydrolysis is not due to the sequence per se but to the structural environment of the 3'-terminus. The enzyme is an exonuclease which initiates hydrolysis at the 3'-terminus and removes 5'-mononucleotides in a random fashion
-
-
-
additional information
?
-
Q38DE2
in vitro, TbRND exhibits 3' to 5' exoribonuclease activity, with specificity toward uridine homopolymers, including the 3' oligo(U) tails of guide RNAs that provide the sequence information for RNA editing, substrate is gA6 RNA
-
-
-
additional information
?
-
Trypanosoma brucei 29-13
Q38DE2
in vitro, TbRND exhibits 3' to 5' exoribonuclease activity, with specificity toward uridine homopolymers, including the 3' oligo(U) tails of guide RNAs that provide the sequence information for RNA editing, substrate is gA6 RNA
-
-
-
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 containing extra residues at the 3'-terminus
5'-mononucleotide + tRNA
show the reaction diagram
-
ribonuclease D is not essential for the normal growth of Escherichia coli or bacteriophage T4 or for the biosynthesis of a T4 suppressor tRNA
-
-
?
tRNA containing extra residues at the 3'-terminus
5'-mononucleotide + tRNA
show the reaction diagram
-
the enzyme can act at the 3'-terminus of tRNA in vivo. RNase D participates in tRNA metabolism
-
-
?
additional information
?
-
-
enzyme is involved in structured RNA processing
-
-
-
additional information
?
-
-
the enzyme may represent an intracellular scavenging mechanism for denatured tRNAs and other inactive RNA molecules
-
-
-
additional information
?
-
Trypanosoma brucei, Trypanosoma brucei 29-13
Q38DE2
in vitro, TbRND exhibits 3' to 5' exoribonuclease activity, with specificity toward uridine homopolymers, including the 3' oligo(U) tails of guide RNAs that provide the sequence information for RNA editing
-
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Co2+
-
enzyme requires a divalent cation for activity. This requirement can be satisfied by Mg2+, Mn2+ or Co2+
K+
-
stimulates
Mg2+
-
required, optimal concentration is 5 mM
Mg2+
-
enzyme requires a divalent cation for activity. This requirement can be satisfied by Mg2+, Mn2+ or Co2+
Mn2+
-
can partially replace for Mg2+
Mn2+
-
enzyme requires a divalent cation for activity. This requirement can be satisfied by Mg2+, Mn2+ or Co2+
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
5,5'-dithiobis(2-nitrobenzoic acid)
-
the enzyme is relatively insensitive to sulfhydryl reagents
EDTA
-
inhibition can be reversed by Mg2+
HgCl2
-
the enzyme is relatively insensitive to sulfhydryl reagents, increased inhibition after addition of dithiothreitol
NEM
-
the enzyme is relatively insensitive to sulfhydryl reagents
p-hydroxymercuribenzoate
-
the enzyme is relatively insensitive to sulfhydryl reagents
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
NH4+
-
stimulates
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
-
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7.5
9
-
with diesterase-treated tRNA as substrate
9.1
9.5
-
-
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
8
10.3
-
pH 8.0: about 50% of maximal activity, pH 10.3: about 70% of maximal activity
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
24
-
Q38DE2
assay at
pI VALUE
pI VALUE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6.2
-
-
electrofocusing, pH-range 2-11
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
Q38DE2
predicted mitochondrial targeting sequence present at the N-terminus of TbRND
Manually annotated by BRENDA team
Trypanosoma brucei 29-13
-
predicted mitochondrial targeting sequence present at the N-terminus of TbRND
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
Escherichia coli (strain K12)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
40000
-
-
gel filtration
60000
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
monomer
-
1 * 40000, SDS-PAGE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
the enzyme is devoid of nucleic acid
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
1.6 A resolution crystal structure. Hanging-drop technique was used for subsequent crystal optimizations. Each drop contains equal volumes of protein and reservoir solution. RNase D protein crystallizes in the presence of 2.02.5 M ammonium sulfate and various buffer conditions. The crystals used here are grown with a reservoir containing 2.22.3 sulfate and 0.1 M HEPES (pH 7.0). Crystals of RNase D usually appeared overnight under these conditions. Crystals grow to full size in 27 days, with a maximum size of over 1 mm in each dimension. Zn 2+ -containing crystals are grown with 1 mM ZnSO4 included in the reservoir prior to drop setup. Mercury(II) derivatives are prepared by soaking RNase D crystals (without Zn2+) overnight in the mother liquor containing 5 mM HgSO4
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
50
-
-
3-4 min, 50% loss of the activity against phosphodiesterase-treated tRNA
additional information
-
-
the enzyme is sensitive to inactivation by elevated temperatures but can be protected by a variety of RNAs
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-20C, 6 months, enzyme retains 90% of its activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant GST- and His-tagged TbRND from Escherichia coli by sequential affinity chromatography on glutathione and nickel affinity resins
Q38DE2
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ORF Tb09.211.3670, DNA and amino acid sequence determination and analysis, generation of pRND-MHT for tetracycline-regulated expression of TbRND with a C-terminal myc-His6-TAP tag in Trypanosoma brucei, expression of N-terminally GST- and His-tagged TbRND in Escherichia coli
Q38DE2
EXPRESSION
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
the expression of mitochondrial exoribonuclease TbRND is highly up-regulated in the insect proliferative stage of the parasite
Q38DE2
the expression of mitochondrial exoribonuclease TbRND is highly up-regulated in the insect proliferative stage of the parasite
Trypanosoma brucei 29-13
-
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D80A
Q38DE2
site-directed mutagenesis
D80A
Trypanosoma brucei 29-13
-
site-directed mutagenesis
-