Information on EC 2.7.7.79 - tRNAHis guanylyltransferase

for references in articles please use BRENDA:EC2.7.7.79
Word Map on EC 2.7.7.79
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:


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

EC NUMBER
COMMENTARY hide
2.7.7.79
-
RECOMMENDED NAME
GeneOntology No.
tRNAHis guanylyltransferase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
App-tRNAHis + GTP = pppGp-tRNAHis + AMP
show the reaction diagram
p-tRNAHis + ATP + GTP = pppGp-tRNAHis + AMP + diphosphate
show the reaction diagram
p-tRNAHis + ATP = App-tRNAHis + diphosphate
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
p-tRNAHis:GTP guanylyltransferase (ATP-hydrolysing)
In eukarya an additional guanosine residue is added post-transcriptionally to the 5'-end of tRNAHis molecules. The addition occurs opposite a universally conserved adenosine73 and is thus the result of a non-templated 3'-5' addition reaction. The additional guanosine residue is an important determinant for aminoacylation by EC 6.1.1.21, histidyl-tRNA ligase. The enzyme requires a divalent cation for activity [2]. ATP activation is not required when the substrate contains a 5'-triphosphate (ppp-tRNAHis) [3].
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
mt-tRNALeuDelta+2 + GTP
? + diphosphate
show the reaction diagram
substrate mimics the presumed in vivo substrate for 5'-end repair, in which the two 5'-mismatched nucleotides have been removed and the resulting tRNA transcript begins with G+3. TLP1 and TLP2 catalyse robust addition of the missing 5'-G nucleotides to this substrate
-
-
?
p-tRNAHis + ATP
App-tRNAHis + diphosphate
show the reaction diagram
p-tRNAHis + ATP + dGTP
?
show the reaction diagram
-
dGTP is almost as efficient as GTP for the guanylylation process. While GDP still serves for the enzymatic reaction, GMP is accepted very poorly by the enzyme
-
-
?
p-tRNAHis + ATP + GDP
?
show the reaction diagram
-
dGTP is almost as efficient as GTP for the guanylylation process. While GDP still serves for the enzymatic reaction, GMP is accepted very poorly by the enzyme
-
-
?
p-tRNAHis + ATP + GTP
pppG-p-tRNAHis + AMP + diphosphate
show the reaction diagram
-
the extra nucleotide in position -1 of mitochondrial and eukaryotic cytoplasmic tRNAHis molecules is added posttranscriptionally to the 5' end of the tRNA by a histidine-tRNA specific guanylyltransferase
-
-
?
p-tRNAHis + ATP + GTP
pppGp-tRNAHis + AMP + diphosphate
show the reaction diagram
p-tRNAHis + dATP + GTP
pppGp-tRNAHis + dAMP + diphosphate
show the reaction diagram
-
30% of the activity compared to ATP
-
-
?
ppp-tRNAHis + GTP
pppGp-tRNAHis + diphosphate
show the reaction diagram
ppp-tRNALeu + GTP
?
show the reaction diagram
the D68A mutation causes a dramatic decrease in the rigorous specificity of Thg1 for tRNAHis. This single alteration enhances the kcat/KM for ppp-tRNALeu by 38-fold relative to that of wild-type Thg1
-
-
?
ppp-tRNAPhe + GTP
?
show the reaction diagram
the D68A mutation causes a dramatic decrease in the rigorous specificity of Thg1 for tRNAHis. This single alteration enhances the kcat/KM for ppp-tRNAPhe by nearly 100-fold relative to that of wild-type Thg1
-
-
?
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
p-tRNAHis + ATP + GTP
pppG-p-tRNAHis + AMP + diphosphate
show the reaction diagram
-
the extra nucleotide in position -1 of mitochondrial and eukaryotic cytoplasmic tRNAHis molecules is added posttranscriptionally to the 5' end of the tRNA by a histidine-tRNA specific guanylyltransferase
-
-
?
p-tRNAHis + ATP + GTP
pppGp-tRNAHis + AMP + diphosphate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
-
the requirement for a divalent metal ion can be satisfied by Mg2+, Mn2+, and to a lesser extent by Co2+ and Zn2+. Activity in presence of 10 mM Co2+ is 13% compared to the activity in presence of 10 mM Mg2+
Mn2+
-
the requirement for a divalent metal ion can be satisfied by Mg2+, Mn2+, and to a lesser extent by Co2+ and Zn2+. Activity in presence of 10 mM Mn2+ is 70% compared to the activity in presence of 10 mM Mg2+
Zn2+
-
the requirement for a divalent metal ion can be satisfied by Mg2+, Mn2+, and to a lesser extent by Co2+ and Zn2+. Activity in presence of 10 mM Zn2+ is 15% compared to the activity in presence of 10 mM Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,5'-dithiobis(2-nitrobenzoic)acid
-
progressive loss of activity with increasing amounts of DTNB. Thus, sulfhydryl groups are involved in maintaining the active state of the enzyme or are involved in the mechanism
ADP
-
20 mM (7fold excess over ATP), 94% inhibition
AMP
-
20 mM (7fold excess over ATP), complete inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0017
p-tRNAHis
-
pH 7.5, 22C
-
0.00016 - 0.00024
ppp-tRNAHis
-
0.0013
ppp-tRNALeu
-
pH 7.5, 22C, mutant enzyme D68A
-
0.00041 - 0.025
ppp-tRNAPhe
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0122
p-tRNAHis
-
pH 7.5, 22C
-
0.00089 - 0.0011
ppp-tRNAHis
-
0.00005
ppp-tRNALeu
-
pH 7.5, 22C, mutant enzyme D68A
-
0.00012 - 0.000172
ppp-tRNAPhe
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
14
mt-tRNALeuDelta+2
pH 7.5, 22C; pH 7.5, 22C
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.3
-
assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
assay at
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28000
-
x * 28000, calculated from sequence
30000
-
x * 30000, SDS-PAGE
58000
-
2 * 58000, SDS-PAGE
120000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 28000, calculated from sequence; x * 30000, SDS-PAGE
dimer
-
2 * 58000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with ATP and with GTP. The enzyme is a tetramer. Catalysis of the 3'-5' reaction with 5'-monophosphorylated tRNA necessitates first an activation step, generating a 5'-adenylylated intermediate prior to a second nucleotidyl transfer step, in which a nucleotide is transferred to the tRNA 5'-end. Distinct binding sites are observed for the nucleotides involved in these two steps
in complex with tRNAHis, ATP and GTP. tRNAHis guanylyltransferase catalyzes reverse (3'-5') nucleotide addition. The directionality of nucleotide polymerization is determined by the orientation of approach of the nucleotide substrate. The tRNA substrate enters the enzyme's active site from the opposite direction (180 degrees flip) compared with similar nucleotide substrates of canonical 5'-3' polymerases, and the finger domains are on opposing sides of the core palm domain. in complexes with ATP and GTP, the adenine base of ATP1 is more deeply embedded into the nucleotide-binding pocket than GTP1 and recognized by hydrogen bonds with the main chain of Asp47, and the side chain of Lys44 interacts directly with ATP1. The base of GTP1 forms hydrogen bonds with the main-chain atoms of Glu43 and Asp47
Q5AFK5
2.3 A crystal structure of human THG1 shares structural homology with canonical 5'-3' DNA polymerases and adenylyl/guanylyl cyclases, two enzyme families known to use a two-metal-ion mechanism for catalysis
-
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme shows a drastically shortened half-life in the absence of divalent metal ions
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
His 6-Thg1p is purified from Escherichia coli
-
recombinant enzyme
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression of His 6-Thg1p in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K43A
the observed rate of adenylylation is decreased by about 10fold, while the rate of guanylylation is virtually unchanged
M158A
no effect on the observed rate of activation
M158N
mutation does not impart the preference for ATP to the enzyme, but it further improves the kinetics for GTP-dependent activation by 10fold relative to the wild-type
K43A
-
the observed rate of adenylylation is decreased by about 10fold, while the rate of guanylylation is virtually unchanged
-
M158A
-
no effect on the observed rate of activation
-
M158N
-
mutation does not impart the preference for ATP to the enzyme, but it further improves the kinetics for GTP-dependent activation by 10fold relative to the wild-type
-
V55A
-
homozygous mutation identified in three siblings who presented with cerebellar signs, developmental delay, dysarthria, and pyramidal signs and had cerebellar atrophy on brain MRI. A defect in the protein tRNAHis guanylyltransferase activity is excluded in vitro
D131A
-
23% of wild-type activity
D153A
-
poorly expressed. D153A is stable, but its expression is somehow toxic to Escherichia coli. 312% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
D47A
-
54.7% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
D68A
-
59% of wild-type G(-1) addition activity. The mutation causes a dramatic decrease in the rigorous specificity of Thg1 for tRNAHis. This single alteration enhances the kcat/KM for ppp-tRNAPhe by nearly 100-fold relative to that of wild-type Thg1. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
D77A
-
less than 0.1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
E13A
-
0.1%-1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
E192A
-
23% of wild-type activity
E75A
-
105% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
E78A
-
less than 0.1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
G74A
-
44.4% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
H155A
-
less than 0.1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
K190A
-
less than 0.1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
K211A
-
0.1%-1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
K44A
-
0.1%-1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
K96A
-
less than 0.1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
N157A
-
23% of wild-type activity
N161A
-
0.1%-1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
N201A
-
less than 0.1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
N46A
-
22.6% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
P45A
-
62.3% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
R133A
-
0.1%-1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
R150A
-
0.1%-1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
R27A
-
0.1%-1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
R93A
-
0.1%-1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
W113A/I156V
-
40.1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
Y146A
-
23% of wild-type activity
Y160A
-
0.1%-1% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
Y202A
-
poorly expressed, The Y202A variant is present at very low levels in soluble crude extracts and yields nearly undetectable levels of protein after purification, suggesting that this alteration leads to lack of overall stability
Y40A
-
42.9% of wild-type G(-1) addition activity. G(-) i.e. the extra nucleotide posttranscriptionally added in position (-1) of tRNA
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
homozygosity for the V55A mutation in THG1L is the cause of an abnormal mitochondrial network in the patient fibroblasts, likely by interfering with THG1L activity towards MFN2
Show AA Sequence (853 entries)
Please use the Sequence Search for a specific query.