Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
IUBMB CommentsIn 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.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
adenosylcobinamide-GDP + diphosphate
GTP + adenosylcobinamide phosphate
GTP + adenosylcobinamide phosphate
diphosphate + adenosylcobinamide-GDP
additional information
?
-
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
-
?
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
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
-
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
-
?
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
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
G8D
mutant non-functional, monomer/dimer ratio: 33/67
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
-
site-directed mutagenesis
H45A/H46A
-
site-directed mutagenesis
H46A
-
site-directed mutagenesis
H46N
-
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
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
Please wait a moment until the data is sorted. This message will disappear when the data is sorted.
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