Information on EC 2.7.7.62 - adenosylcobinamide-phosphate guanylyltransferase

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

EC NUMBER
COMMENTARY hide
2.7.7.62
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RECOMMENDED NAME
GeneOntology No.
adenosylcobinamide-phosphate guanylyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
GTP + adenosylcobinamide phosphate = diphosphate + adenosylcobinamide-GDP
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
adenosylcobalamin biosynthesis from cobyrinate a,c-diamide I
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adenosylcobalamin biosynthesis from cobyrinate a,c-diamide II
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adenosylcobalamin salvage from cobinamide I
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adenosylcobalamin salvage from cobinamide II
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Metabolic pathways
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Porphyrin and chlorophyll metabolism
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vitamin B12 metabolism
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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.
CAS REGISTRY NUMBER
COMMENTARY hide
169592-55-6
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
adenosylcobinamide-GDP + diphosphate
GTP + adenosylcobinamide phosphate
show the reaction diagram
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
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
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
show the reaction diagram
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.102
5'-deoxyadenosylcobinamide phosphate
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pH 8.8, 37C
0.0184
adenosylcobinamide phosphate
apparent KM
0.0024 - 0.0213
GTP
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.093
5'-deoxyadenosylcobinamide phosphate
Salmonella enterica subsp. enterica serovar Typhimurium
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pH 8.8, 37C
4.7
adenosylcobinamide phosphate
Methanocaldococcus jannaschii
Q58517
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0.058 - 1.8
GTP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00000002
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GTP:guanylyltransferase specific activity, mutant H46A
0.00000003
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GTP:guanylyltransferase specific activity, mutant H46N
0.000205
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GTP:guanylyltransferase specific activity, mutant H45A
0.00067
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GTP:guanylyltransferase specific activity, wild-type
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
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activity assay
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5 - 9.5
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50% activity retains in this range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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activity assay
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 60
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retains 50% activity in this range, temperatures above 60C results in drastic decrease of activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.3
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predicted pI for the denatured protein
7.2
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experimetally determined isoelectric point
7.5
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experimetally determined isoelectric point
PDB
SCOP
CATH
ORGANISM
UNIPROT
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
Methanocaldococcus jannaschii (strain ATCC 43067 / DSM 2661 / JAL-1 / JCM 10045 / NBRC 100440)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
19700
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predicted mass, based on DNA sequence
20000
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amino acid sequence, small polypeptide with 180 amino acids
21600
monomer, determined by gel filtation and SDS-PAGE
34000
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SDS-PAGE
43000
dimer, determined by gel filtation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
trimer
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
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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
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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
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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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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80
MjCobY denaturates at 80C with a concomitant loss of activity
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
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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
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644435
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, stored under a N2 headspace, retains 98% of its activity for at least 3 weeks
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cell-free extracts are prepared
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in two steps by hydrophobic interaction and ion-exchange chromatography
single-step method based on hydrophobic interaction chromatography
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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
expressed in Escherichia coli
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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
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into the vector pBAD24 for expression in serovar Typhimurium strain JE8268
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into the vector pT7-7 for expression in Escherichia coli BL21-CodonPlus DE3 cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D101G
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in vitro activity relative to wild-type: 71%, monomer/dimer ratio: 34/66
D83G
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in vitro activity relative to wild-type: 100%, monomer/dimer ratio: 82/18
E18D
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in vitro activity relative to wild-type: 56%, monomer/dimer ratio: 40/60
G8D
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mutant non-functional, monomer/dimer ratio: 33/67
I144D/F146D/Y163R
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monomer/dimer/aggregate ratio: 77/23/0. Specific activity (pmol/min/microgram): 156 (monomer), 49 (dimer)
K19A
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in vitro activity relative to wild-type: 16%, monomer/dimer ratio: 36/64
L38D/K41D
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monomer/dimer/aggregate ratio: 53/25/22. Specific activity (pmol/min/microgram): 125 (monomer), 120 (dimer), 78 (aggregate)
L38K/K41D
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monomer/dimer/aggregate ratio: 52/26/22. Specific activity (pmol/min/microgram): 124 (monomer), 119 (dimer), 85 (aggregate)
N177R
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in vitro activity relative to wild-type: 5%, monomer/dimer ratio: not determined
N179D
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in vitro activity relative to wild-type: 95%, monomer/dimer ratio: not determined
P20A
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in vitro activity relative to wild-type: 93%, monomer/dimer ratio: 63/37
P54A
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in vitro activity relative to wild-type: 62%, monomer/dimer ratio: 50/50
P94A
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in vitro activity relative to wild-type: 43%, monomer/dimer ratio: 52/48
R13A
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in vitro activity relative to wild-type: 15%, monomer/dimer ratio: 14/86
S100A
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in vitro activity relative to wild-type: 100%, monomer/dimer ratio: not determined
S50A
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in vitro activity relative to wild-type: 100%, monomer/dimer ratio: not determined
T180A
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in vitro activity relative to wild-type: 73%, monomer/dimer ratio: not determined
T53S
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in vitro activity relative to wild-type: 84%, monomer/dimer ratio: 76/24
T56A
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in vitro activity relative to wild-type: 102%, monomer/dimer ratio: not determined
Y80F
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in vitro activity relative to wild-type: 65%, monomer/dimer ratio: 74/26
D83G
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in vitro activity relative to wild-type: 100%, monomer/dimer ratio: 82/18
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G153D
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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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K19A
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in vitro activity relative to wild-type: 16%, monomer/dimer ratio: 36/64
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S100A
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in vitro activity relative to wild-type: 100%, monomer/dimer ratio: not determined
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T180A
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in vitro activity relative to wild-type: 73%, monomer/dimer ratio: not determined
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Y80F
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in vitro activity relative to wild-type: 65%, monomer/dimer ratio: 74/26
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H45A/H46A
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site-directed mutagenesis
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