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Information on EC 2.7.7.62 - adenosylcobinamide-phosphate guanylyltransferase
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2.7.7.62 adenosylcobinamide-phosphate guanylyltransferaseIUBMB Comments
In Salmonella typhimurium LT2, under anaerobic conditions, CobU (EC 2.7.7.62 and EC 2.7.1.156), CobT (EC 2.4.2.21), CobC (EC 3.1.3.73) and CobS (EC 2.7.8.26) catalyse reactions in the nucleotide loop assembly pathway, which convert adenosylcobinamide (AdoCbi) into adenosylcobalamin (AdoCbl). CobT and CobC are involved in 5,6-dimethylbenzimidazole activation whereby 5,6-dimethylbenzimidazole is converted to its riboside, alpha-ribazole. The second branch of the nuclotide loop assembly pathway is the cobinamide (Cbi) activation branch where AdoCbi or adenosylcobinamide-phosphate is converted to the activated intermediate AdoCbi-GDP by the bifunctional enzyme Cob U. The final step in adenosylcobalamin biosynthesis is the condensation of AdoCbi-GDP with alpha-ribazole, which is catalysed by EC 2.7.8.26, cobalamin synthase (CobS), to yield adenosylcobalamin. CobU is a bifunctional enzyme that has both kinase (EC 2.7.1.156) and guanylyltransferase (EC 2.7.7.62) activities. However, both activities are not required at all times.The kinase activity has been proposed to function only when S. typhimurium is assimilating cobinamide whereas the guanylyltransferase activity is required for both assimilation of exogenous cobinamide and for de novo synthesis of adenosylcobalamin . The guanylyltransferase reaction is a two-stage reaction with formation of a CobU-GMP intermediate . Guanylylation takes place at histidine-46.
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Word Map
- 2.7.7.62
- mannose-1-phosphate
- gdp-mannose
-
guanylylation
- adenosylcobalamin
- reesei
- trichoderma
- dolichyl
-
actinidia
- phosphomannomutase
-
nonorthologous
-
adocbl
- gdp-l-galactose
-
nucleotidyltransferase
- galactomannan
-
shuman
- l-galactose
-
methanocaldococcus
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
guanyltransferase, mjcoby, gtp:adenosylcobinamide-phosphate guanylyltransferase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
adenosylcobinamide kinase
adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase
adenosylcobinamide phosphate guanyltransferase
-
-
adenosylcobinamide phosphate guanylyltransferase
AdoCbi kinase/AdoCbi-phosphate guanylyltransferase
-
-
-
-
CobP
-
bifunctional enzyme with kinase and guanylyltransferase activity
CobU
CobY
GTP: adenosylcobinamide-phosphate guanylyltransferase
-
GTP: AdoCbi-P guanylyltransferase
GTP:adenosylcobinamide-phosphate guanylyltransferase
guanyltransferase
MjCobY
adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase
-
-
-
-
adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase
-
bifunctional enzyme
adenosylcobinamide phosphate guanylyltransferase
-
-
CobU
-
-
-
-
CobU
-
-
CobU
-
bifunctional enzyme adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase
GTP:adenosylcobinamide-phosphate guanylyltransferase
-
-
-
-
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
GTP + adenosylcobinamide phosphate = diphosphate + adenosylcobinamide-GDP
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
BRENDA
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SYSTEMATIC NAME
IUBMB Comments
GTP:adenosylcobinamide-phosphate guanylyltransferase
In Salmonella typhimurium LT2, under anaerobic conditions, CobU (EC 2.7.7.62 and EC 2.7.1.156), CobT (EC 2.4.2.21), CobC (EC 3.1.3.73) and CobS (EC 2.7.8.26) catalyse reactions in the nucleotide loop assembly pathway, which convert adenosylcobinamide (AdoCbi) into adenosylcobalamin (AdoCbl). CobT and CobC are involved in 5,6-dimethylbenzimidazole activation whereby 5,6-dimethylbenzimidazole is converted to its riboside, alpha-ribazole. The second branch of the nuclotide loop assembly pathway is the cobinamide (Cbi) activation branch where AdoCbi or adenosylcobinamide-phosphate is converted to the activated intermediate AdoCbi-GDP by the bifunctional enzyme Cob U. The final step in adenosylcobalamin biosynthesis is the condensation of AdoCbi-GDP with alpha-ribazole, which is catalysed by EC 2.7.8.26, cobalamin synthase (CobS), to yield adenosylcobalamin. CobU is a bifunctional enzyme that has both kinase (EC 2.7.1.156) and guanylyltransferase (EC 2.7.7.62) activities. However, both activities are not required at all times.The kinase activity has been proposed to function only when S. typhimurium is assimilating cobinamide whereas the guanylyltransferase activity is required for both assimilation of exogenous cobinamide and for de novo synthesis of adenosylcobalamin [4]. The guanylyltransferase reaction is a two-stage reaction with formation of a CobU-GMP intermediate [1]. Guanylylation takes place at histidine-46.
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CAS REGISTRY NUMBER
COMMENTARY
169592-55-6
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
adenosylcobinamide-GDP + diphosphate
GTP + adenosylcobinamide phosphate
-
over-expressing cell extracts, 2 mM ATP, 5 mM MgCl2, 16 h, pH 8, 37°C
reaction stop by 25 mM KCN and 10 min heating at 80°C, analysis by mass spectrometry
-
?
adenosylcobinamide-GDP + diphosphate
GTP + adenosylcobinamide phosphate
-
2 mM ATP, 5 mM MgCl2, 16 h, pH 8, 37°C
reaction stop by 25 mM KCN and 10 min heating at 80°C, analysis by mass spectrometry
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12, uses GTP to guanylate itself, then transfers this GMP group to AdoCbi-P to yield AdoCbi-GDP, the final product of the reaction
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
late steps of cobalamin biosynthesis, involved in de novo synthesis of vitamin B12, nucleotide loop assembly, required for assimilation of exogenous cobinamide and de novo synthesis of adenosylcobalamin, reaction proceeds via a covalently modified CobU-GMP intermediate
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
assembly of the nucleotide loop of cobalamin
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
biosynthesis of cobalamin, enzyme catalyzes the phosphorylation of the 1-amino-O-2-propanol side chain of the adenosylcobinamide ring
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
the bifunctional enzyme adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase is involved in the biosynthesis of cobalamin
-
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 2.7.1.156 and guanyltransferase EC 2.7.7.62 activities
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 2.7.1.156 and guanyltransferase EC 2.7.7.62 activities, cobY gene encodes a protein with GTP:AdoCbi-P guanyltransferase activity but no NTP:AdoCbi kinase activity
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 2.7.1.156 and guanyltransferase EC 2.7.7.62 activities, cobY gene encodes a protein with GTP:AdoCbi-P guanyltransferase activity but no NTP:AdoCbi kinase activity
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 2.7.1.156 and guanyltransferase EC 2.7.7.62 activities
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 2.7.1.156 and guanyltransferase EC 2.7.7.62 activities
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 2.7.1.156 and guanyltransferase EC 2.7.7.62 activities
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 2.7.1.156 and guanyltransferase EC 2.7.7.62 activities
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 2.7.1.156 and guanyltransferase EC 2.7.7.62 activities
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 2.7.1.156 and guanyltransferase EC 2.7.7.62 activities
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 2.7.1.156 and guanyltransferase EC 2.7.7.62 activities
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 27.1.1.156 and guanyltransferase EC 2.7.7.62 activities
-
?
additional information
?
-
-
CobU is a bifunctional enzyme that has both kinase EC 27.1.1.156 and guanyltransferase EC 2.7.7.62 activities, ATP does not serve as an efficient substrate for the CobU transferase activity, CobU does not have any detectable ATP:adenosyltransferase activity
-
?
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12, uses GTP to guanylate itself, then transfers this GMP group to AdoCbi-P to yield AdoCbi-GDP, the final product of the reaction
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
-
late steps of cobalamin biosynthesis, involved in de novo synthesis of vitamin B12, nucleotide loop assembly, required for assimilation of exogenous cobinamide and de novo synthesis of adenosylcobalamin, reaction proceeds via a covalently modified CobU-GMP intermediate
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
assembly of the nucleotide loop of cobalamin
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
biosynthesis of cobalamin, enzyme catalyzes the phosphorylation of the 1-amino-O-2-propanol side chain of the adenosylcobinamide ring
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
-
cobalamin biosynthesis, involved in de novo synthesis of vitamin B12
-
?
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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DISEASE
TITLE OF PUBLICATION
LINK TO PUBMED
Tuberculosis
Structure and mutational analysis of the archaeal GTP:AdoCbi-P guanylyltransferase (CobY) from Methanocaldococcus jannaschii: insights into GTP binding and dimerization.
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.102
5'-deoxyadenosylcobinamide phosphate
-
pH 8.8, 37°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.093
5'-deoxyadenosylcobinamide phosphate
-
pH 8.8, 37°C
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.00000002
-
GTP:guanylyltransferase specific activity, mutant H46A
0.00000003
-
GTP:guanylyltransferase specific activity, mutant H46N
0.000205
-
GTP:guanylyltransferase specific activity, mutant H45A
0.00067
-
GTP:guanylyltransferase specific activity, wild-type
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
8.5 - 9.5
-
50% activity retains in this range
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25 - 60
-
retains 50% activity in this range, temperatures above 60°C results in drastic decrease of activity
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5.3
-
predicted pI for the denatured protein
7.2
-
experimetally determined isoelectric point
7.5
-
experimetally determined isoelectric point
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
brenda
Show AA Sequence and Transmembrane Helices (21196 entries)
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
19700
-
predicted mass, based on DNA sequence
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
monomer
trimer
dimer
MjCobYWT is comprised of a population of dimers and monomers. CobY is active in both the monomeric and dimeric forms
dimer
-
MjCobYWT is comprised of a population of dimers and monomers. CobY is active in both the monomeric and dimeric forms
-
monomer
MjCobYWT is comprised of a population of dimers and monomers. CobY is active in both the monomeric and dimeric forms
monomer
wild-type CobY is monomeric in solution in both its apo- and GTP-bound formssolution structural studies
monomer
-
MjCobYWT is comprised of a population of dimers and monomers. CobY is active in both the monomeric and dimeric forms
-
monomer
-
wild-type CobY is monomeric in solution in both its apo- and GTP-bound formssolution structural studies
-
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
the solution structure has a mixed alpha/beta fold consisting of seven beta-strands and five alpha-helices, very similar to a Rossmann fold. Titration of apo-CobY with GTP results in large changes in amide proton chemical shifts. The CobY:GTP complex is unstable over time, GTP hydrolyzes and the protein converts slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, yields NMR spectra similar to those of wild-type CobY in both its apo-state and in complex with GTP. The CobYG153D:GTP complex is also unstable over time
three-dimensional crystal structure of the guanylyltransferase (CobY) enzyme (mutant G153D) from Methanocaldococcus jannaschii (MjCobY)1 in complex with GTP at 2.8 A resolution is reported
X-ray crystal structure determined by molecular replacement to 2.2 A resolution, crystals grown by the batch method, cocrystallized with GTP and diphosphate, crystals belong to space group P2(1)2(1)2(1) with unit cell dimensions a : 58.4 A, b : 87.8 A, and c : 101.6 A
-
X-ray structure determined to 2.3 A resolution, crystals grown by microbatch, enzyme is crystallized in space group C222(1) with unit cell dimensions of a : 96.4 A, b : 114.4 A, and c : 106.7 A
-
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D101G
in vitro activity relative to wild-type: 71%, monomer/dimer ratio: 34/66
D83G
in vitro activity relative to wild-type: 100%, monomer/dimer ratio: 82/18
E18D
in vitro activity relative to wild-type: 56%, monomer/dimer ratio: 40/60
G153D
I144D/F146D/Y163R
monomer/dimer/aggregate ratio: 77/23/0. Specific activity (pmol/min/microgram): 156 (monomer), 49 (dimer)
K19A
in vitro activity relative to wild-type: 16%, monomer/dimer ratio: 36/64
L38D/K41D
monomer/dimer/aggregate ratio: 53/25/22. Specific activity (pmol/min/microgram): 125 (monomer), 120 (dimer), 78 (aggregate)
L38K/K41D
monomer/dimer/aggregate ratio: 52/26/22. Specific activity (pmol/min/microgram): 124 (monomer), 119 (dimer), 85 (aggregate)
N177R
in vitro activity relative to wild-type: 5%, monomer/dimer ratio: not determined
N179D
in vitro activity relative to wild-type: 95%, monomer/dimer ratio: not determined
P20A
in vitro activity relative to wild-type: 93%, monomer/dimer ratio: 63/37
P54A
in vitro activity relative to wild-type: 62%, monomer/dimer ratio: 50/50
P94A
in vitro activity relative to wild-type: 43%, monomer/dimer ratio: 52/48
R13A
in vitro activity relative to wild-type: 15%, monomer/dimer ratio: 14/86
S100A
in vitro activity relative to wild-type: 100%, monomer/dimer ratio: not determined
S50A
in vitro activity relative to wild-type: 100%, monomer/dimer ratio: not determined
T180A
in vitro activity relative to wild-type: 73%, monomer/dimer ratio: not determined
T53S
in vitro activity relative to wild-type: 84%, monomer/dimer ratio: 76/24
T56A
in vitro activity relative to wild-type: 102%, monomer/dimer ratio: not determined
Y80F
in vitro activity relative to wild-type: 65%, monomer/dimer ratio: 74/26
D83G
-
in vitro activity relative to wild-type: 100%, monomer/dimer ratio: 82/18
-
G153D
-
residues R13 and K19 of the mutant directly coordinate the phosphate group of GTP in the X-ray structure. The X-ray structure of the CobYG153D:GTP complex is modeled as a homodimer
-
K19A
-
in vitro activity relative to wild-type: 16%, monomer/dimer ratio: 36/64
-
S100A
-
in vitro activity relative to wild-type: 100%, monomer/dimer ratio: not determined
-
T180A
-
in vitro activity relative to wild-type: 73%, monomer/dimer ratio: not determined
-
Y80F
-
in vitro activity relative to wild-type: 65%, monomer/dimer ratio: 74/26
-
H45A/H46A
-
site-directed mutagenesis
G153D
crystal structure of mutant is determined at 2.8 A: The protein crystallizes in the hexagonal space group P31 with 4 chains in the asymmetric unit. Mutant shows reduced activity and is composed of a monomer. Results from isothermal titration calorimetry experiments show that MjCobYG153D has 10fold higher affinity for GTP than wild-type, but fails to bind the corrinoid substrate
G153D
in vitro activity relative to wild-type: 2%, monomer/dimer ratio: 54/46
G153D
residues R13 and K19 of the mutant directly coordinate the phosphate group of GTP in the X-ray structure. The X-ray structure of the CobYG153D:GTP complex is modeled as a homodimer
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
80
MjCobY denaturates at 80°C with a concomitant loss of activity
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
no soluble N-terminal His-tagged protein detectable after immobilized metal affinity chromatography from over-expressing Salmonella enterica serovar Typhimurium LT2
-
substrates provides little protection from inhibition by DTNB, GTP provides slight protection from inhibition of the guanylyltransferase activity
-
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
in presence of oxygen guanylyltransferase activity is lost
-
644435
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, stored under a N2 headspace, retains 98% of its activity for at least 3 weeks
-
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in two steps by hydrophobic interaction and ion-exchange chromatography
single-step method based on hydrophobic interaction chromatography
-
tandem hydrophobic interaction chromatography on two HiTrap Phenyl FastFlow columns, dialysation, and dye ligand chromatography on HiTrap Blue HP column (elution with 2 M NaCl)
-
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
in pET-16b (pCLK740) for expression with N-terminal deca-His-tag in Salmonella enterica serovar Typhimurium LT2, strain JE8407 lacking endogenous CobU and YcfN
-
into the vector pBAD24 for expression in serovar Typhimurium strain JE8268
-
into the vector pT7-7 for expression in Escherichia coli BL21-CodonPlus DE3 cells
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Warren, M.J.; Raux, E.; Schubert, H.L.; Escalante-Semerena, J.C.
The biosynthesis of adenosylcobalamin (vitamin B12)
Nat. Prod. Rep.
19
390-412
2002
Priestia megaterium, Escherichia coli, Salmonella enterica, Methanothermobacter thermautotrophicus, Propionibacterium freudenreichii, Pseudomonas denitrificans (nom. rej.), Methanothermobacter thermautotrophicus DELTAH
brenda
O'Toole, G.A.; Escalante-Semerena, J.C.
Purification and characterization of the bifunctional CobU enzyme of Salmonella typhimurium LT2. Evidence for a CobU-GMP intermediate
J. Biol. Chem.
270
23560-23569
1995
Salmonella enterica subsp. enterica serovar Typhimurium
brenda
Thompson, T.B.; Thomas, M.G.; Escalante-Semerena, J.C.; Rayment, I.
Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase from Salmonella typhimurium determined to 2.3 resolution
Biochemistry
37
7686-7695
1998
Salmonella enterica subsp. enterica serovar Typhimurium
brenda
Thompson, T.B.; Thomas, M.G.; Escalante-Semerena, J.C.; Rayment, I.
Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) complexed with GMP: evidence for a substrate-induced transferase active site
Biochemistry
38
12995-13005
1999
Salmonella enterica subsp. enterica serovar Typhimurium
brenda
Thomas, M.G.; Thompson, T.B.; Rayment, I.; Escalante-Semerena, J.C.
Analysis of the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU) enzyme of Salmonella typhimurium LT2. Identification of residue His-46 as the site of guanylylation
J. Biol. Chem.
275
27576-27586
2000
Salmonella enterica subsp. enterica serovar Typhimurium
brenda
O'Toole, G.A.; Escalante-Semerena, J.C.
cobU-dependent assimilation of nonadenosylated cobinamide in cobA mutants of Salmonella typhimurium
J. Bacteriol.
175
6328-6336
1993
Salmonella enterica subsp. enterica serovar Typhimurium
brenda
Hsu, F.C.; Ho, T.J.; Lai, C.C.; Lin, C.F.; Chen, H.P.
Cloning, sequencing, expression, and single-step purification of the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU) from Salmonella typhimurium ATCC 19585
Protein Expr. Purif.
42
178-181
2005
Salmonella enterica subsp. enterica serovar Typhimurium
brenda
Otte, M.M.; Woodson, J.D.; Escalante-Semerena, J.C.
The thiamine kinase (YcfN) enzyme plays a minor but significant role in cobinamide salvaging in Salmonella enterica
J. Bacteriol.
189
7310-7315
2007
Escherichia coli, Salmonella enterica
brenda
Gray, M.J.; Tavares, N.K.; Escalante-Semerena, J.C.
The genome of Rhodobacter sphaeroides strain 2.4.1 encodes functional cobinamide salvaging systems of archaeal and bacterial origins
Mol. Microbiol.
70
824-836
2008
Cereibacter sphaeroides
brenda
Otte, M.M.; Escalante-Semerena, J.C.
Biochemical characterization of the GTP:adenosylcobinamide-phosphate guanylyltransferase (CobY) enzyme of the hyperthermophilic archaeon Methanocaldococcus jannaschii
Biochemistry
48
5882-5889
2009
Methanocaldococcus jannaschii (Q58517), Methanocaldococcus jannaschii
brenda
Newmister, S.; Otte, M.; Escalante-Semerena, J.; Rayment, I.
Structure and Mutational Analysis of the Archaeal GTP:AdoCbi-P Guanylyltransferase (CobY) from Methanocaldococcus jannaschii: Insights into GTP Binding and Dimerization
Biochemistry
50
5301-5313
2011
Methanocaldococcus jannaschii (Q58517), Methanocaldococcus jannaschii, Methanocaldococcus jannaschii DSM 2661 (Q58517)
brenda
Singarapu, K.K.; Otte, M.M.; Tonelli, M.; Westler, W.M.; Escalante-Semerena, J.C.; Markley, J.L.
Solution structural studies of GTP:adenosylcobinamide-phosphateguanylyl transferase (CobY) from Methanocaldococcus jannaschii
PLoS ONE
10
e0141297
2015
Methanocaldococcus jannaschii (Q58517), Methanocaldococcus jannaschii DSM 2661 (Q58517)
brenda
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