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Information on EC 2.5.1.17 - corrinoid adenosyltransferase and Organism(s) Homo sapiens and UniProt Accession Q96EY8

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IUBMB Comments
The corrinoid adenosylation pathway comprises three steps: (i) reduction of Co(III) within the corrinoid to Co(II) by a one-electron transfer. This can occur non-enzymically in the presence of dihydroflavin nucleotides or reduced flavoproteins . (ii) Co(II) is bound by corrinoid adenosyltransferase, resulting in displacement of the lower axial ligand by an aromatic residue. The reduction potential of the 4-coordinate Co(II) intermediate is raised by ~250 mV compared with the free compound, bringing it to within physiological range. This is followed by a second single-electron transfer from either free dihydroflavins or the reduced flavin cofactor of flavoproteins, resulting in reduction to Co(I) . (iii) the Co(I) conducts a nucleophilic attack on the adenosyl moiety of ATP, resulting in transfer of the deoxyadenosyl group and oxidation of the cobalt atom to Co(III) state. Three types of corrinoid adenosyltransferases, not related by sequence, have been described. In the anaerobic bacterium Salmonella enterica they are encoded by the cobA gene (a housekeeping enzyme involved in both the de novo biosynthesis and the salvage of adenosylcobalamin), the pduO gene (involved in (S)-propane-1,2-diol utilization), and the eutT gene (involved in ethanolamine utilization). Since EutT hydrolyses triphosphate to diphosphate and phosphate during catalysis, it is classified as a separate enzyme. The mammalian enzyme belongs to the PduO type. The enzyme can act on other corrinoids, such as cob(II)inamide.
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Homo sapiens
UNIPROT: Q96EY8
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The taxonomic range for the selected organisms is: Homo sapiens
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Reaction Schemes
hide(Overall reactions are displayed. Show all >>)
2
ATP
+
2
cob(II)alamin
+
a reduced flavoprotein
=
2
triphosphate
+
2
adenosylcob(III)alamin
+
an oxidized flavoprotein
2
+
2
+
a reduced flavoprotein
=
2
+
2
+
an oxidized flavoprotein
2
ATP
+
2
cob(II)yrinic acid a,c-diamide
+
a reduced flavoprotein
=
2
triphosphate
+
2
adenosylcob(III)yrinic acid a,c-diamide
+
an oxidized flavoprotein
2
+
2
+
a reduced flavoprotein
=
2
+
2
+
an oxidized flavoprotein
Synonyms
atp:cob(i)alamin adenosyltransferase, cob(i)alamin adenosyltransferase, lrpduo, atp:co(i)rrinoid adenosyltransferase, atp:cobalamin adenosyltransferase, st2180, ta1434, st1454, atp:corrinoid adenosyltransferase, cobalamin adenosyltransferase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ATP:cobalamin adenosyltransferase
-
human adenosyltransferase
-
human ATP: cob(I)alamin adenosyltransferase
PduO-type enzyme, MMAB gene
adenosyltransferase, vitamin B12s
-
-
-
-
aquacob(I)alamin adenosyltransferase
-
-
-
-
aquocob(I)alamin adenosyltransferase
-
-
-
-
ATP:cob(I)alamin adenosyltransferase
-
-
ATP:cob(I)alamin Cobeta-adenosyltransferase
-
-
-
-
ATP:cob(I)alamin transferase (ATR)
-
-
-
-
ATP:cobalamin adenosyltransferase
-
-
ATP:corrinoid adenosyltransferase
-
-
-
-
cob(I)alamin adenosyltransferase
-
-
-
-
cob(I)yrinic acid a,c-diamide adenosyltransferase
-
-
-
-
CobA
-
-
-
-
hATR
-
-
methylmalonic aciduria type B
-
-
MMAB
-
-
MMAB protein
-
-
PduO-type corrinoid adenosyltransferase
-
-
vitamin B12s adenosyltransferase
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
adenosyl group transfer
-
adenosyl group transfer
-
-
-
-
PATHWAY SOURCE
PATHWAYS
SYSTEMATIC NAME
IUBMB Comments
ATP:cob(II)alamin Cobeta-adenosyltransferase
The corrinoid adenosylation pathway comprises three steps: (i) reduction of Co(III) within the corrinoid to Co(II) by a one-electron transfer. This can occur non-enzymically in the presence of dihydroflavin nucleotides or reduced flavoproteins [3]. (ii) Co(II) is bound by corrinoid adenosyltransferase, resulting in displacement of the lower axial ligand by an aromatic residue. The reduction potential of the 4-coordinate Co(II) intermediate is raised by ~250 mV compared with the free compound, bringing it to within physiological range. This is followed by a second single-electron transfer from either free dihydroflavins or the reduced flavin cofactor of flavoproteins, resulting in reduction to Co(I) [7]. (iii) the Co(I) conducts a nucleophilic attack on the adenosyl moiety of ATP, resulting in transfer of the deoxyadenosyl group and oxidation of the cobalt atom to Co(III) state. Three types of corrinoid adenosyltransferases, not related by sequence, have been described. In the anaerobic bacterium Salmonella enterica they are encoded by the cobA gene (a housekeeping enzyme involved in both the de novo biosynthesis and the salvage of adenosylcobalamin), the pduO gene (involved in (S)-propane-1,2-diol utilization), and the eutT gene (involved in ethanolamine utilization). Since EutT hydrolyses triphosphate to diphosphate and phosphate during catalysis, it is classified as a separate enzyme. The mammalian enzyme belongs to the PduO type. The enzyme can act on other corrinoids, such as cob(II)inamide.
CAS REGISTRY NUMBER
COMMENTARY hide
37277-84-2
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
?
ATP + hydroxycobalamin
adenosylcobalamin + phosphate + diphosphate
show the reaction diagram
37°C, pH 8, 0.5 mM ATP, 0.05 mM hydroxycobalamin, in presence of 1 mM titanium(III)citrate
measured by decrease in absorbance at 388 nm
-
?
cob(II)alamin
cob(I)alamin
show the reaction diagram
G97, T161, and H183 possible role in stabilizing four-coordinate, cob(II)alamin C-terminal His-tagged enzyme binds cob(II)alamin base-off while N-terminal His-tagged enzyme binds it base-on (impaired base-off transition), only mutants S68F, K78Q, K78R, R186W, and R190C also bind cob(II)alamin base-off
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
ATP + cob(I)alamin
triphosphate + adenosylcobalamin
show the reaction diagram
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
CTP + cob(I)alamin
triphosphate + cytosylcobalamin
show the reaction diagram
-
polymorphic variants 239K and 239M, 9% activity compared to ATP with enzyme variant 239K, 6% activity compared to ATP with enzyme variant 239M
-
-
?
GTP + cob(I)alamin
triphosphate + guanosylcobalamin
show the reaction diagram
-
polymorphic variants 239K and 239M, 16% activity compared to ATP with enzyme variant 239K, 14% activity compared to ATP with enzyme variant 239M
-
-
?
UTP + cob(I)alamin
triphosphate + uridylcobalamin
show the reaction diagram
-
polymorphic variants 239K and 239M, 8% activity compared to ATP with enzyme variant 239K, 6% activity compared to ATP with enzyme variant 239M
-
-
?
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
-
adenosyltransferase enzymes lower the thermodynamic barrier of the Co2+ toCo+ reduction needed for the formation of the unique organometalic Co-C bond of adenosylcobalamin
-
?
ATP + cob(I)alamin
triphosphate + adenosylcobalamin
show the reaction diagram
-
polymorphic variants 239K and 239M, biosynthesis of adenosylcobalamin
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
twenty residues at the enzyme’s N-terminus become ordered upon binding of ATP to form an ATP-binding site, structure, overview
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
-
induces formation of activated Co2+ species
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00287 - 0.215
ATP
0.00158 - 0.0302
cob(I)alamin
0.0063 - 0.32
ATP
0.00037 - 0.06
cob(I)alamin
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
19.2 - 297.6
ATP
19.2 - 297.6
cob(I)alamin
0.011 - 0.037
cob(I)alamin
additional information
ATP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.061
0.098
0.026
-
reductant: PduS protein, specific activity of hATR using different electron sources for the adenosylation of cob(II)alamin
0.027
-
purified recombinant GST-tagged mutant R191W
0.072
-
reductant: NADPH-dependent ferredoxin protein reductase, specific activity of hATR using different electron sources for the adenosylation of cob(II)alamin
0.089
-
purified recombinant GST-tagged wild-type enzyme
0.19
-
recombinant enzyme variant 239M
0.22
-
recombinant enzyme variant 239K
0.25
-
reductant: dihydroflavin, specific activity of hATR using different electron sources for the adenosylation of cob(II)alamin
0.49
-
reductant: Ti(III)citrate, specific activity of hATR using different electron sources for the adenosylation of cob(II)alamin
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
assay at
37
-
assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
Uniprot
Manually annotated by BRENDA team
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
mRNA expression detected
Manually annotated by BRENDA team
skin fibroblasts show low abundance
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
Arg19 in the mitochondrial targeting sequence is important
Manually annotated by BRENDA team
from skin cells
-
Manually annotated by BRENDA team
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
MMAB_HUMAN
250
0
27388
Swiss-Prot
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
52000
SDS-PAGE, recombinant enzyme without mitochondrial targeting sequence
55000
calculated from amino acid sequence of recombinant enzyme without mitochondrial targeting sequence
56000
SDS-PAGE, recombinant enzyme with mitochondrial targeting sequence
58000
calculated from amino acid sequence of recombinant enzyme with mitochondrial targeting sequence
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
trimer
the enzyme forms a tightly associated trimer, where the monomer comprises a five-helix bundle and the active sites lie on the subunit interfaces, invariant residues and their function within the ATR structure, detailed overview
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C119Y
wild-type kinetics, decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression, no rescue of ATR-deficient Salmonella strain BE620
C189Y
inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620
D218N
substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
D64G
substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
D90N
inactive in vitro (10fold excess of substrate compared to standard)
E193K
inactive in vitro (10fold excess of substrate compared to standard), conserved residue, mutation found in methylmalonic aciduria patients
E84K
inactive in vitro (10fold excess of substrate compared to standard)
E91K
inactive in vitro (10fold excess of substrate compared to standard)
F212S
inactive in vitro (10fold excess of substrate compared to standard)
F83S
large change in KM for ATP and cob(I)alamin, F83 has direct contact with ATP
G63E
substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
G87R
inactive in vitro (10fold excess of substrate compared to standard)
G97E
wild-type kinetics, mutation distant from proposed active site, no rescue of ATR-deficient Salmonella strain BE620 possibly due to impaired reduction of cob(II)alamin to cob(I)alamin, expressed at wild-type levels
G97R
substantially reduced Vmax, mutation distant from proposed active site, no rescue of ATR-deficient Salmonella strain BE620
H183Y
wild-type kinetics, mutation distant from proposed active site, no rescue of ATR-deficient Salmonella strain BE620 possibly due to impaired reduction of cob(II)alamin to cob(I)alamin, expressed at wild-type levels
K78Q
inactive in vitro (10fold excess of substrate compared to standard)
K78R
substantially reduced Vmax, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
L220P
inactive in vitro (10fold excess of substrate compared to standard)
L223P
inactive in vitro (10fold excess of substrate compared to standard)
L92S
inactive in vitro (10fold excess of substrate compared to standard)
R186W
inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620, conserved residue, mutation found in methylmalonic aciduria patients
R190C
inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620, conserved residue, mutation found in methylmalonic aciduria patients
R191W
inactive in vitro (10fold excess of substrate compared to standard), decreased adenosylcobalamin production in vivo, impaired protein folding leads to degradation and, thus, low expression (but can be purified), no rescue of ATR-deficient Salmonella strain BE620, conserved residue, mutation found in methylmalonic aciduria patients
R194G
inactive in vitro (10fold excess of substrate compared to standard)
R215K
inactive in vitro (10fold excess of substrate compared to standard), lack of activity in vitro, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
R225K
lack of activity in vitro, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
R76G
large change in KM for ATP and cob(I)alamin, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
S126L
large change in KM for ATP and cob(I)alamin, decreased adenosylcobalamin production in vivo partly corrected by increased hydroxycobalamin concentration, part of proposed active site, role in ATP/cobalamin binding, no rescue of ATR-deficient Salmonella strain BE620
S217R
inactive in vitro (10fold excess of substrate compared to standard)
S68F
substantially reduced Vmax, residue S68 has role in ATP-binding
S94L
inactive in vitro (10fold excess of substrate compared to standard)
T161I
wild-type kinetics, decreased adenosylcobalamin production in vivo but rescues ATR-deficient Salmonella strain BE620 possibly due to impaired reduction of cob(II)alamin to cob(I)alamin, expressed at wild-type levels
E193K
-
site-directed mutagenesis, the mutant enzyme is not expressed
R186W
-
catalytically inactive patient mutation leading to the inherited disorder methylmalonic aciduria. Mutant is examined using intrinsic fluorescence quenching of MMAB as a measure of ligand-binding. R190H and R186W significantly disrupt the affinity between MMAB and adenosylcobalmin. Arg 186 and Arg-190 may be critical for the transfer of the 5'-deoxyadenosyl group from ATP to cob(I)alamin, possibly by contributing to the precise positioning of the two substrates to permit catalysis to occur
R190H
-
catalytically inactive patient mutation leading to the inherited disorder methylmalonic aciduria. Mutant is examined using intrinsic fluorescence quenching of MMAB as a measure of ligand-binding. R190H and R186W significantly disrupt the affinity between MMAB and adenosylcobalmin. Arg 186 and Arg-190 may be critical for the transfer of the 5'-deoxyadenosyl group from ATP to cob(I)alamin, possibly by contributing to the precise positioning of the two substrates to permit catalysis to occur
R191W/A135T
-
site-directed mutagenesis, the mutant enzyme shows 30% reduced activity compared to the wild-type enzyme
additional information
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
after expression at 16°C, lysis with French pressure cell, pH 7.5, and centrifugation
centrifugation and SDS-PAGE
2 recombinant polymorphic variants 239K and 239M of the enzyme from Escherichia coli in a multistep procedure to homogeneity, 4.5- and 5.4fold, respectively
-
recombinant maltose-binding-protein or GST fusion wild-type and mutant enzymes from Escherichia coli strain DH5alpha by amylose and glutathione affinity chromatography, respectively
-
using a HisTrap FF column
-
using a HiTrap SP HP FPLC column and gel filtration
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
without mitochondrial targeting sequence from plasmid pNL166 harbouring the coding sequence, native or with C-terminal octa-His tag into pTA925 to introduce a linker region necessary for expression in Escherichia coli BL21DE3 RIL and ATR-deficient Salmonella strain BE620, hATR in pCF13 (N-terminal octa-His tag) and pCF15 (C-terminal octa-His tag) for transformation and random mutagenesis in Escherichia coli mutator strain XL1-Red followed by expression in BE620
DNA sequence determination and analysis, expression of 2 polymorphic variants 239K and 239M of the enzyme in Escherichia coli as soluble proteins
-
expressed in Escherichia coli
-
expressed in Escherichia coli as an N-terminal His-tagged fusion protein
-
expression of the enzyme in C57/Bl6 mice using an adeno-associated virus vector, rAAV 2 and 8 vectors, with primer pairs specific to the cytomegalovirus enhancer/chicken beta-actin, CBAT, promoter, quantitative and semiquantitative expression analysis in murine liver, overview
-
expression of wild-type and mutant enzymes in Escherichia coli strain DH5alpha as maltose-binding-protein or GST fusion protein
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
insight in molecular mechanism of methylmalonic aciduria
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Leal, N.A.; Park, S.D.; Kima, P.E.; Bobik, T.A.
Identification of the human and bovine ATP:Cob(I)alamin adenosyltransferase cDNAs based on complementation of a bacterial mutant
J. Biol. Chem.
278
9227-9234
2003
Bos taurus, Homo sapiens (Q96EY8), Homo sapiens
Manually annotated by BRENDA team
Stich, T.A.; Yamanishi, M.; Banerjee, R.; Brunold, T.C.
Spectroscopic evidence for the formation of a four-coordinate Co2+ cobalamin species upon binding to the human ATP:cobalamin adenosyltransferase
J. Am. Chem. Soc.
127
7660-7661
2005
Homo sapiens
Manually annotated by BRENDA team
Leal, N.A.; Olteanu, H.; Banerjee, R.; Bobik, T.A.
Human ATP:cob(I)alamin adenosyltransferase and its interaction with methionine synthase reductase
J. Biol. Chem.
279
47536-47542
2004
Homo sapiens
Manually annotated by BRENDA team
Schubert, H.L.; Hill, C.P.
Structure of ATP-bound human ATP:cobalamin adenosyltransferase
Biochemistry
45
15188-15196
2006
Homo sapiens (Q96EY8), Homo sapiens
Manually annotated by BRENDA team
Erger, K.E.; Conlon, T.J.; Leal, N.A.; Zori, R.; Bobik, T.A.; Flotte, T.R.
In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8
J. Gene Med.
9
462-469
2007
Homo sapiens
Manually annotated by BRENDA team
Zhang, J.; Dobson, C.M.; Wu, X.; Lerner-Ellis, J.; Rosenblatt, D.S.; Gravel, R.A.
Impact of cblB mutations on the function of ATP:cob(I)alamin adenosyltransferase in disorders of vitamin B12 metabolism
Mol. Genet. Metab.
87
315-322
2006
Homo sapiens
Manually annotated by BRENDA team
Fan, C.; Bobik, T.A.
Functional characterization and mutation analysis of human ATP:Cob(I)alamin adenosyltransferase
Biochemistry
47
2806-2813
2008
Homo sapiens (Q96EY8), Homo sapiens
Manually annotated by BRENDA team
Mera, P.; Escalante-Semerena, J.
Dihydroflavin-driven adenosylation of 4-coordinate Co(II) corrinoids: are cobalamin reductases enzymes or electron transfer proteins?
J. Biol. Chem.
285
2911-2917
2010
Homo sapiens, Limosilactobacillus reuteri
Manually annotated by BRENDA team
Zhang, J.; Wu, X.; Padovani, D.; Schubert, H.; Gravel, R.
Ligand-binding by catalytically inactive mutants of the cblB complementation group defective in human ATP:cob(I)alamin adenosyltransferase
Mol. Genet. Metab.
98
278-284
2009
Homo sapiens
Manually annotated by BRENDA team