Information on EC 2.5.1.17 - cob(I)yrinic acid a,c-diamide adenosyltransferase

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

EC NUMBER
COMMENTARY
2.5.1.17
-
RECOMMENDED NAME
GeneOntology No.
cob(I)yrinic acid a,c-diamide adenosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ATP + cob(I)yrinic acid a,c-diamide = triphosphate + adenosylcob(III)yrinic acid a,c-diamide
show the reaction diagram
-
-
-
-
ATP + cob(I)yrinic acid a,c-diamide = triphosphate + adenosylcob(III)yrinic acid a,c-diamide
show the reaction diagram
reaction mechanism
-
ATP + cob(I)yrinic acid a,c-diamide = triphosphate + adenosylcob(III)yrinic acid a,c-diamide
show the reaction diagram
Cys79, Cys80, and Cys83 are important for catalysis
-
ATP + cob(I)yrinic acid a,c-diamide = triphosphate + adenosylcob(III)yrinic acid a,c-diamide
show the reaction diagram
active site and substrate binding sites and structures, molecular architecture
-
ATP + cob(I)yrinic acid a,c-diamide = triphosphate + adenosylcob(III)yrinic acid a,c-diamide
show the reaction diagram
not yet confimed
Q970Z7, -
ATP + cob(I)yrinic acid a,c-diamide = triphosphate + adenosylcob(III)yrinic acid a,c-diamide
show the reaction diagram
not yet confimed
-
ATP + cob(I)yrinic acid a,c-diamide = triphosphate + adenosylcob(III)yrinic acid a,c-diamide
show the reaction diagram
residues Phe91 and Trp93 play a critical role in the mechanism of four-coordinate Cob(II)alamin formation in the active site of the Salmonella enterica ATP:Co(I)rrinoid adenosyltransferase enzyme, overview.. The enzyme adopts a closed conformation and residues Phe91 and Trp93 displace 5,6-dimethylbenzimidazole, the lower nucleotide ligand base of cobalamin, to generate a transient four-coordinate cobalamin, which is critical in the formation of the AdoCbl Co-C bond, important role of bulky hydrophobic side chains in the active site. CobA and PduO increase the redox potential of the cob(II)alamin/cob(I)alamin couple to facilitate formation of the Co-C bond, in both cases the polar coordination of the lower ligand to the cobalt ion is eliminated by placing that face of the corrin ring adjacent to a cluster of bulky hydrophobic side chains
-
ATP + cob(I)yrinic acid a,c-diamide = triphosphate + adenosylcob(III)yrinic acid a,c-diamide
show the reaction diagram
active site and substrate binding sites and structures, molecular architecture
Lactobacillus reuteri CRL1098
-
-
ATP + cobinamide = triphosphate + adenosylcobinamide
show the reaction diagram
-
-
-
-
ATP + cobinamide = triphosphate + adenosylcobinamide
show the reaction diagram
reaction mechanism, substrate structure
-
ATP + cobinamide = triphosphate + adenosylcobinamide
show the reaction diagram
reaction mechanism
-
ATP + cobinamide = triphosphate + adenosylcobinamide
show the reaction diagram
Cys79, Cys80, and Cys83 are important for catalysis
-
ATP + cobinamide = triphosphate + adenosylcobinamide
show the reaction diagram
active site and substrate binding sites and structures, molecular architecture
-
ATP + cobinamide = triphosphate + adenosylcobinamide
show the reaction diagram
nucleophilic attack from reduced Co1+ ion of cob(I)alamin to the C-5 carbon of ATP, ordered substrate binding mechanism with ATP being first and essential for catalysis
-
ATP + cobinamide = triphosphate + adenosylcobinamide
show the reaction diagram
residues Phe91 and Trp93 play a critical role in the mechanism of four-coordinate Cob(II)alamin formation in the active site of the Salmonella enterica ATP:Co(I)rrinoid adenosyltransferase enzyme, overview. The enzyme adopts a closed conformation and residues Phe91 and Trp93 displace 5,6-dimethylbenzimidazole, the lower nucleotide ligand base of cobalamin, to generate a transient four-coordinate cobalamin, which is critical in the formation of the AdoCbl Co-C bond, important role of bulky hydrophobic side chains in the active site. CobA and PduO increase the redox potential of the cob(II)alamin/cob(I)alamin couple to facilitate formation of the Co-C bond, in both cases the polar coordination of the lower ligand to the cobalt ion is eliminated by placing that face of the corrin ring adjacent to a cluster of bulky hydrophobic side chains
-
ATP + cobinamide = triphosphate + adenosylcobinamide
show the reaction diagram
to overcome the thermodynamically challenging Co2+ -> Co1+ reduction, the enzyme drastically weakens the axial ligand-Co2+ bond so as to generate effectively four-coordinate Co2+-corrinoid species, mechanism, overview. The entire hydrophobic pocket below the corrin ring, and not just residue F112, is critical for the removal of the axial ligand from the cobalt center of the Co2+-corrinoids. Large role of the ATP-induced active-site conformational changes with respect to the formation of 4c Co(II)Cbl
-
ATP + cobinamide = triphosphate + adenosylcobinamide
show the reaction diagram
active site and substrate binding sites and structures, molecular architecture
Lactobacillus reuteri CRL1098
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
adenosyl group transfer
-
-
-
-
adenosyl group transfer
Q9HIA7
-
adenosyl group transfer
Q970Z7, -
number of ion pairs around the putative active site (i.e., Glu125) may correlate with adenosyl group transfer onto cob(I)alamin
adenosyl group transfer
-
-
adenosyl group transfer
-
-
adenosyl group transfer
-
-
PATHWAY
KEGG Link
MetaCyc Link
adenosylcobalamin biosynthesis from cobyrinate a,c-diamide I
-
adenosylcobalamin biosynthesis from cobyrinate a,c-diamide II
-
adenosylcobalamin salvage from cobalamin
-
adenosylcobalamin salvage from cobinamide I
-
adenosylcobalamin salvage from cobinamide II
-
Metabolic pathways
-
Porphyrin and chlorophyll metabolism
-
SYSTEMATIC NAME
IUBMB Comments
ATP:cob(I)yrinic acid-a,c-diamide Cobeta-adenosyltransferase
The corrinoid adenosylation pathway comprises three steps: (i) reduction of Co(III) to Co(II) by a one-electron transfer. This can be carried out by EC 1.16.1.3, aquacobalamin reductase or non-enzymically in the presence of dihydroflavin nucleotides [2]. (ii) Co(II) is reduced to Co(I) in a second single-electron transfer by EC 1.16.1.4, cob(II)alamin reductase and (iii) the Co(I) conducts a nucleophilic attack on the adenosyl moiety of ATP to leave the cobalt atom in a Co(III) state (EC 2.5.1.17). The enzyme responsible for the adenosylation reaction is the product of the gene cobO in the aerobic bacterium Pseudomonas denitrificans and of the gene cobA in the anaerobic bacterium Salmonella typhimurium. In P. denitrificans, the enzyme shows specificity for cobyrinic acid a,c-diamide and the corrinoids that occur later in the biosynthetic pathway whereas CobA seems to have broader specificity [3]. While CobA has a preference for ATP and Mn2+, it is able to transfer a variety of nucleosides to the cobalt, including CTP, UTP and GTP, in decreasing order of preference [4] and to use Mg2+ instead of Mn2+.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
adenosyltransferase, vitamin B12s
-
-
-
-
aquacob(I)alamin adenosyltransferase
-
-
-
-
aquocob(I)alamin adenosyltransferase
-
-
-
-
ATP: cobalamin adenosyltransferase
Q2SZ09
-
ATP:co(I)rrinoid adenosyltransferase
-
-
ATP:co(I)rrinoid adenosyltransferase
P31570
-
ATP:cob(I)alamin adenosyltransferase
-
-
ATP:cob(I)alamin adenosyltransferase
-
-
ATP:cob(I)alamin adenosyltransferase
Q970Z7
-
ATP:cob(I)alamin adenosyltransferase
Sulfolobus tokodaii 7
Q970Z7
-
-
ATP:cob(I)alamin Cobeta-adenosyltransferase
-
-
-
-
ATP:cob(I)alamin transferase (ATR)
-
-
-
-
ATP:cobalamin adenosyltransferase
-
-
ATP:cobalamin adenosyltransferase
Q9HIA7
-
ATP:corrinoid adenosyltransferase
-
-
-
-
ATP:corrinoid adenosyltransferase
-
-
ATP:corrinoid adenosyltransferase
P31570
-
cob(I)alamin adenosyltransferase
-
-
-
-
CobA
-
-
-
-
CobA
-
gene name
CobA-type ATP:Co(I)rrinoid adenosyltransferase
-
-
cobalamin adenosyltransferase
-
-
cobalamin adenosyltransferase
P31570
-
cobO mutant SVQ336
-
-
cobO mutant SVQ524
-
-
cobO mutantSVQ336
-
-
hATR
-
-
hATR
Q96EY8
-
homologeous to PduO-type ATP: cob(I)alamin adenosyltransferase
-
-
human adenosyltransferase
Q96EY8
-
human ATP: cob(I)alamin adenosyltransferase
Q96EY8
PduO-type enzyme, MMAB gene
human-type ACA
-
-
human-type ATP: co(I)rrinoid adenosyltransferase
-
PduO
human-type ATP: cob(I)alamin adenosyltransferase
-
-
LrPduO
-
PduO from Lactobacillus reuteri
methylmalonic aciduria type B
-
-
MMAB
-
-
MMAB protein
-
-
PduO
-
gene name
PduO
-
gene name
PduO
Lactobacillus reuteri CRL1098
-
-
-
PduO
-
gene name
PduO adenosyltransferase
-
-
PduO enzyme
-
encoded by pduO gene
PduO-type ACA
-
-
PduO-type adenosine 5'-triphosphate:corrinoid adenosyltransferase
-
-
PduO-type ATP: cobalamin adenosyltransferase
Q2SZ09
-
PduO-type ATP:Co(I)rrinoid adenosyltransferase
-
-
PduO-type ATP:Co(I)rrinoid adenosyltransferase
Lactobacillus reuteri CRL1098
-
-
-
PduO-type ATP:cob(I)alamin adenosyltransferase
-
-
PduO-type ATP:corrinoid adenosyltransferase
-
-
PduO-type corrinoid adenosyltransferase
-
-
ST1454
Q970Z7
locus name
ST1454
Sulfolobus tokodaii 7
Q970Z7
locus name
-
vitamin B12s adenosyltransferase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
37277-84-2
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
gene pduO
-
-
Manually annotated by BRENDA team
2 common polymorphic variants 239K and 239M of the enzyme
-
-
Manually annotated by BRENDA team
gene cblB
-
-
Manually annotated by BRENDA team
strain CRL1098, gene pduO
-
-
Manually annotated by BRENDA team
Lactobacillus reuteri CRL1098
strain CRL1098, gene pduO
-
-
Manually annotated by BRENDA team
strain Go1, orf MM3138, housekeeping gene cobAMm
-
-
Manually annotated by BRENDA team
recombinant strain SC510 Rifr containing plasmid pXL227, this DNA seuqence encodes for several enzymes
SwissProt
Manually annotated by BRENDA team
EutT; 2 enzymes encoded by genes eutT and cobA with different primary sequences, cobA is a housekeeping gene, several derivatives of strain TR6583, several geno- and phenotypes, overview
-
-
Manually annotated by BRENDA team
gene cobA
-
-
Manually annotated by BRENDA team
gene eutT, eutT is no housekeeping gene in contrast to the other enzyme-encoding gene cobA
-
-
Manually annotated by BRENDA team
serovar typhimurium
-
-
Manually annotated by BRENDA team
sv. typhimurium, several strains, overview, gene cobA
-
-
Manually annotated by BRENDA team
CobA; 2 enzymes encoded by genes eutT and cobA with different primary sequences, cobA is a housekeeping gene, several derivatives of strain TR6583, several geno- and phenotypes, overview
SwissProt
Manually annotated by BRENDA team
serovar typhimurium, structural gene pduO, this DNA sequence encodes for several enzymes
SwissProt
Manually annotated by BRENDA team
HH103, strain SVQ336; HH103, strain SVQ524
-
-
Manually annotated by BRENDA team
thermophilic archeon
-
-
Manually annotated by BRENDA team
Sulfolobus tokodaii 7
-
SwissProt
Manually annotated by BRENDA team
; gene TA1434
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
evolution
-
cob(I)alamin adenosyltransferases are separated into three type groups according to their amino acid sequences: CobA, PduO, and EutT. Among these adenosyltransferases, the PduO-type adenosyltransferases are the most widely distributed enzyme type. Whereas the CobA-type enzyme, which is constitutively expressed, is encoded by the cobA gene, PduO and EutT-type adenosyltransferases are encoded within large operons whose functions are required for the catabolism of 1,2-propanediol or ethanolamine. Despite the fact that all three families of adenosyltransferases catalyze the same overall reaction, they share little sequence identity of below 20% and the CobA and PduO enzymes have fairly different three-dimensional structures
evolution
-
ATP:co(I)rrinoid adenosyltransferases, ACATs, are separated into three families namely, CobA, EutT, and PduO
physiological function
-
ATP:co(I)rrinoid adenosyltransferases are enzymes that catalyze the formation of adenosylcobalamin, i.e. coenzyme B12, from cobalamin and ATP. CobA increases the redox potential of the cob(II)alamin/cob(I)alamin couple to facilitate formation of the Co-C bond. In Salmonella enterica, CobA is the housekeeping enzyme that is required for de novo AdoCbl synthesis and for salvaging incomplete precursors and cobalamin from the environment
malfunction
-
a HH103 cobO mutant (strain SVQ524), is constructed by the insertion of omega interposon in the BglII site of cobO gene. Mutant is auxotroph for methionine and cobalamin as it is shown that the presence of either compound restores the growth on minimal medium. Mutant SVQ524 fails to nodulate on Vigna radiate but is able to nodulate on Glycine max cvs. Williams and Peking and Cajanus cajan. The roots of mutant plants do not secrete enough cobalamin and/or methionine to support growth of cobalamin/methionine auxotrophs in the rhizosphere. The phenotype of SVQ524 is rescued by the addition of methionine or cobalamin to the plant growth media or by the presence of a copy of the cobO gene; a methionine and cobalamin mutant strain (SVQ336) of Sinorhizobium fredii HH103 is obtained by Tn5-lacZ mutagenesis. Sequence analysis show that the transposon is inserted into a gene homologous to cobO. cobO mutant SVQ336 is auxotroph for methionine and cobalamin as it is shown that the presence of either compound restores the growth of SVQ336 on minimal medium
additional information
-
structural comparisons between apo BcPduO and BcPduO in complex with MgATP reveal that the N-terminal strands of both structures are ordered, which is in contrast with the most previously available PduO-type adenosyltransferase structures. Apo BcPduO is bound to additional dioxane molecules causing a side chain conformational change at the Tyr30 residue, which is an important residue that mediates hydrogen bonding with ATP molecules upon binding of cobalamin to the active site
additional information
-
residues Phe91 and Trp93 play a critical role in the mechanism of four-coordinate Cob(II)alamin formation in the active site of the Salmonella enterica ATP:Co(I)rrinoid adenosyltransferase enzyme, overview. The enzyme adopts a closed conformation and residues Phe91 and Trp93 displace 5,6-dimethylbenzimidazole, the lower nucleotide ligand base of cobalamin, to generate a transient four-coordinate cobalamin, which is critical in the formation of the AdoCbl Co-C bond, important role of bulky hydrophobic side chains in the active site. CobA increases the redox potential of the cob(II)alamin/cob(I)alamin couple to facilitate formation of the Co-C bond
additional information
-
active-site region of wild-type and mutant PduOs complexed with Co(II)Cbl and cosubstrate ATP, structure determination and modeling, overview
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2'-deoxy-ATP + cob(I)alamin + H2O
?
show the reaction diagram
-
-
-
-
?
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
Q96EY8
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
P29930, -
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
?
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
?
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
?
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
?
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
?
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
r
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
?
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
P29930, -
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
?
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
P31570
-
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
?
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
stereospecific process which proceeds with overall inversion of configuration at C-5' of the adenosyl moiety
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
transfers the adenosyl-group of ATP to the reduced cobalt atom of the cobalamin molecule
adenosylcobalamin, B12-coenzyme or deoxyadenosyl-B12
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
P31570
the main role of this enzyme is apparently the conversion of inactive cobalamins to adenosyl cobalamin for 1,2 propanediol degradation
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
involved in vitamin B12 metabolism
-
-
-
ATP + cob(I)alamin
triphosphate + adenosylcobalamin
show the reaction diagram
Q9HIA7
-
-
-
-
ATP + cob(I)alamin
triphosphate + adenosylcobalamin
show the reaction diagram
Q9HIA7, -
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcobalamin
show the reaction diagram
-
polymorphic variants 239K and 239M, biosynthesis of adenosylcobalamin, polymorphic variants 239K and 239M, ATP is highly preferred as adenosyl donor
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
-
-, adenosyltransferase enzymes lower the thermodynamic barrier of the Co2+ to Co+ reduction needed for the formation of the unique organometalic Co-C bond of adenosylcobalamin
-
?
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 + adenosylcob(III)alamin
show the reaction diagram
-
final step in the conversion of vitamin B12 to coenzyme B12, the latter being required for degradation of 1,2-propanediol
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
last step in the corrinoid adenosylation pathway, EutT and CobA
-
-
ir
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
P31570
last step in the corrinoid adenosylation pathway, EutT and CobA
-
-
ir
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
mechanism of adenosylcobalamin biosynthesis
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
CobA has an ATP-binding P-loop motif, while EutT has a cysteine-rich region reminiscent of a conserved S-adenosylmethionine Fe-S cluster motif
-
-
ir
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
P31570
CobA has an ATP-binding P-loop motif, while EutT has a cysteine-rich region reminiscent of a conserved S-adenosylmethionine Fe-S cluster motif
-
-
ir
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
formation of the the essential C-Co bond by transferring the adenosyl group from a molecule of ATP to a transient Co1+ corrinoid species generated by the active site
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
reaction mechanism: assimilated cobalamin is reduced to co(II)alamin, that then binds to the enzyme-ATP complex, further reduction yields a nucleophilic four coordinated Co1+ intermediate that attacks the 5'-carbon of the cosubstrate ATP to generate adenosylcobalamin and triphosphate, spectroscopic analysis, enzyme-induced base-on/base-off conversion activating the cobalamin substrate for reduction
-
-
ir
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
binding of the substrate ATP to ATR that is fully loaded with 5'-deoxyadenosylcobalamin leads to the ejection of 1 equivalent of the cofactor into solution. In the presence of methylmalonyl-CoA mutase and ATP, 5'-deoxyadenosylcobalamin is transferred from ATR to the acceptor protein in a process that exhibits an 3.5fold lower Kact for ATP compared to the one in which cofactor is released into solution. ATP favorably influences cofactor transfer in the forward direction by reducing the ratio of apo-methylmalonyl-CoA mutase/holo-ATR required for delivery of 1 equivalent of 5'-deoxyadenosylcobalamin, from 4 to 1. A rotary rotary mechanism for ATR function is proposed in which, at any given time, only two of its active sites are used for 5'-deoxyadenosylcobalamin synthesis and where binding of ATP to the vacant site leads to the transfer of the high value 5'-deoxyadenosylcobalamin product to the acceptor mutase
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(I)alamine
show the reaction diagram
-
-
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(I)alamine
show the reaction diagram
-
-
-
-
?
ATP + cob(I)alamin
tripolyphosphate + coenzyme B12
show the reaction diagram
Q970Z7, -
-
-
-
?
ATP + cob(I)alamin
tripolyphosphate + coenzyme B12
show the reaction diagram
Sulfolobus tokodaii 7
Q970Z7
-
-
-
?
ATP + cob(I)alamin + H2O
?
show the reaction diagram
Q970Z7, -
anaerobic, 20 min, 80C, pH 8, in presence of 1 mM titanium (III) citrate
ATP-dependent cob(I)alamin consumption to a yet unknown compound
-
?
ATP + cob(I)alamin + H2O
adenosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
37C
monitoring adenosylcobalamin formation at 388 nm in continous spectrophotometric assay
-
?
ATP + cob(I)alamin + H2O
phosphate + diphosphate + adenosylcobalamin
show the reaction diagram
Q970Z7, -
anaerobic, 20 min, 80C, pH 8, in presence of 1 mM titanium (III) citrate
measured by decrease in light absorbance by cob(I)alamin at 388 nm and increase of light absorbance by presumably adenosylcobalamin at 525 nm
-
?
ATP + cobalamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
enzyme is absolutely specific for ATP or dATP as adenosyl donors, ATP is the preferred adenosyl donor
-
-
?
ATP + cobalamin
triphosphate + adenosylcobalamin
show the reaction diagram
Q9HIA7, -
enzyme is absolutely specific for ATP or dATP as adenosyl donors
-
-
ir
ATP + cobinamide
triphosphate + adenosylcobinamide
show the reaction diagram
-
-
-
-
?
ATP + hydroxycobalamin
adenosylcobalamin + phosphate + diphosphate
show the reaction diagram
-
37C, 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
-
?
ATP + hydroxycobalamin
tripolyphosphate + adenosylcobalamin + H2O
show the reaction diagram
-
coenzyme B12 synthesis from vitamin B12, dimethylbenzimidazole arm of vitamin B12 plays no role in substrate positioning, corrinoid adenosylation assay: anaerobic, pH 6, 25C, 1 or 2 mM FMN, 10 or 20 mM NADH, NAD(P)H: flavin oxidoreductase, 2 h incubation for complete reduction of hydroxycobalamin to cob(II)alamin before initiation of adenosyltransfer
measuring difference in absorbance by adenosylcobalamin at 525 nm
-
?
cob(I)alamin + ADP
adenosylcobalamin + diphosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + ADP
adenosylcobalamin + diphosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
adenosylation of the corrinoid ring of cob(I)alamin generates coenzyme B12, i.e. adenosylcobalamin or AdoCbl, an essential cofactor used by enzymes that catalyze intramolecular rearrangements, deaminations, dehydrations, reductions, and reductive dehalogenations
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
adenosylcobalamin is a cofactor required by the methylmalonyl-CoA mutase
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
cobalamin assimilation and recycling pathway, overview, enzyme deficiency causes methylmalonic aciduria, MMA, is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000fold greater than normal, overview
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
the enzyme is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
adenosylation of the corrinoid ring of cob(I)alamin, active site structure, ATP binding motif at the protein N terminus
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
cobalamin is a better substrate than cobinamide, the beta-phosphate of ATP is required for binding to the enzyme
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
in vitro reduced flavodoxin provides an electron to generate the co(I)rrinoid substrate in the CobA active site, modeling of enzyme ligand interaction, residues R9 and R165 are important for CobA-FldA docking but not to catalysis, overview
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
dissociation constant (Kd) of wild-type MMAB for hydroxomethylcobalamin is 0.051 mM and for ATP is 0.365 mM, cobalamin enhances the affinity of MMAB for ATP, while ATP does not show detectable effects on cobalamin binding
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
upon binding to LrPduO that is preincubated with ATP, both Co2+corrinoids undergo a partial (40-50%) conversion to distinct paramagnetic Co2+ species. The spectroscopic signatures of these species are consistent with essentially four-coordinate, square-planar Co2+ complexes. For effecting Co2+ to Co1+ reduction the formation of an activated Co2+ corrinoid intermediate that lacks any significant axial bonding interactions is involved to stabilize the redoxactive, Co 3dz2-based molecular orbital
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
Lactobacillus reuteri CRL1098
-
adenosylation of the corrinoid ring of cob(I)alamin generates coenzyme B12, i.e. adenosylcobalamin or AdoCbl, an essential cofactor used by enzymes that catalyze intramolecular rearrangements, deaminations, dehydrations, reductions, and reductive dehalogenations, adenosylation of the corrinoid ring of cob(I)alamin, active site structure, ATP binding motif at the protein N terminus
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + ?
show the reaction diagram
-
in the co+ assay the cobalt ion of cobalamin is chemically reduced in solution to cob(I)alamin by Ti(III)citrate, allowing the cob(I)alamin adenosylation reaction to be measured directly. The Co+ assay is performed under anoxic conditions
-
-
?
cob(I)alamin + ATP + H2O + H+
adenosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-, FMN and NADH are used to reduce cob(III)alamin to cob(I)alamin, the enzyme shows ATP hydrolyzing activity to adenosine and triphosphate in absence of cob(I)alamin
-
-
?
cob(I)alamin + CTP + H2O + H+
cytosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + dATP + H2O + H+
deoxyadenosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + GTP + H2O + H+
guanosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)alamin + ITP
hypoxanthosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)alamin + UTP
uranylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)inamide + ATP
5'-deoxy-5'-adenosyl-cob(I)inamide + polyphosphate
show the reaction diagram
P29930, -
-
-
-
cob(I)inamide + ATP
adenosylcobinamide + triphosphate
show the reaction diagram
-
-, cobalamin is a better substrate than cobinamide, the beta-phosphate of ATP is required for binding to the enzyme
-
-
?
cob(I)inamide + ATP
adenosylcobinamide + triphosphate
show the reaction diagram
-
upon binding to LrPduO that is preincubated with ATP, both Co2+corrinoids undergo a partial (40-50%) conversion to distinct paramagnetic Co2+ species. The spectroscopic signatures of these species are consistent with essentially four-coordinate, square-planar Co2+ complexes. For effecting Co2+ to Co1+ reduction the formation of an activated Co2+ corrinoid intermediate that lacks any significant axial bonding interactions is involved to stabilize the redoxactive, Co 3dz2-based molecular orbital
-
-
?
cob(I)yric acid + ATP
5'-deoxy-5'-adenosyl-cob(I)yric acid + polyphosphate
show the reaction diagram
P29930, -
-
-
-
cob(I)yrinic acid a,c-diamide + ATP
5'-deoxy-5'-adenosyl-cob(I)yrinic acid a,c-diamide + polyphosphate
show the reaction diagram
P29930, -
-
-
-
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
-
-
?
cob(II)alamin + ATP
adenosylcobalamin + ?
show the reaction diagram
-
in the Co2+ assay the NADPH-dependent flavodoxin protein reductase/flavodoxin system is used to reduce Co2+ to Co+. In this Co2+ assay, the PduO enzyme must bind cob(II)alamin and facilitate the generation of cob(I)alamin in its active site
-
-
?
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
-
-
?
CTP + cob(I)alamin + H2O
cytidylcobalamin + diphosphate + phosphate
show the reaction diagram
-
37C
-
-
?
cyanocob(I)alamin + ATP
tripolyphosphate + alpha-(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide
show the reaction diagram
-
-
-
-
cyanocob(I)alamin + ATP
tripolyphosphate + alpha-(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide
show the reaction diagram
-
-
-
-
-
dATP + cob(I)alamin
tripolyphosphate + deoxyadenosylcobalamin
show the reaction diagram
Q9HIA7
-
-
-
-
dATP + cob(I)alamin
tripolyphosphate + deoxyadenosylcobalamin
show the reaction diagram
Q9HIA7, -
-
-
?
dATP + cobalamin
triphosphate + deoxyadenosylcob(III)alamin
show the reaction diagram
-
enzyme is absolutely specific for ATP or dATP as adenosyl donors, dATP results in 21% of the activity with ATP
-
-
?
dATP + cobalamin
triphosphate + deoxyadenosylcobalamin
show the reaction diagram
Q9HIA7, -
enzyme is absolutely specific for ATP or dATP as adenosyl donors
-
-
ir
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
-
-
?
GTP + cob(I)alamin + H2O
guanosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
37C
-
-
?
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
-
-
?
UTP + cob(I)alamin + H2O
uridylcobalamin + diphosphate + phosphate
show the reaction diagram
-
37C
-
-
?
ITP + cob(I)alamin + H2O
inosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
37C
-
-
?
additional information
?
-
-
-
-
-
-
additional information
?
-
-
-
-
-
-
additional information
?
-
-
UTP, GTP, ITP are poor substrates
-
-
-
additional information
?
-
-
S-adenosylmethionine, vitamin B12r or B12a, AMP, ADP are no substrates
-
-
-
additional information
?
-
-
GTP, CTP show only small activity as substrates
-
-
-
additional information
?
-
-
hydroxocobamides or cyanocobamides in which benzimidazole replaces 5,6-dimethylbenzimidazole, or cyanocobamide in which an adenine group replaces 5,6-dimethylbenzimidazole, can also act as substrates
-
-
-
additional information
?
-
P29930, -
cobyrinic acid is not a substrate
-
-
-
additional information
?
-
-
crucial role of the 2'-OH group of the ribosyl moiety of ATP, the gamma-phosphate of ATP is critical for positioning the target for nucleophilic attack, differences in the base of the nucleotide have no effect on enzyme activity, 2'-deoxynucleotides fails to serve as substrates
-
-
-
additional information
?
-
-
EutT is necessary and sufficient for growth of a cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source
-
-
-
additional information
?
-
P31570
EutT is necessary and sufficient for growth of a cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source
-
-
-
additional information
?
-
-
the enzyme interacts with the methionine synthase reductase MSR, which catalyzes the reduction of cob(II)almin to cob(I)alamin, both enzymes activate each other, stoichiometry of the MSR-ATR system, overview
-
-
-
additional information
?
-
-
the flavodoxin in vivo reducing agent that serves as the electron donor to the enzyme possesses a reduction potential that is considerably more positive than that of the Co2+/1+ couple of the corrinoid substrate, the enzyme overcomes this challenge by formation of an ATP-activated unique paramagnetic Co2+ corrinoid species by partial conversion, overview
-
-
-
additional information
?
-
Q9HIA7, -
conserved residues are R119, R124, and E126
-
-
-
additional information
?
-
-
enzyme-substrate interactions, intermediate/transition structures, spectroscopic mechanism analysis, computational modeling, overview
-
-
-
additional information
?
-
-
no activity with ADP and AMP
-
-
-
additional information
?
-
-
random or ordered ternary complex mechanism, the N-terminal domain is responsible for the catalytic activity and substrate binding and has similar biochemical properties and kinetic constants as the full-length enzyme
-
-
-
additional information
?
-
-
enzyme defects cause methylmalonic aciduria type B, regulation, overview
-
-
-
additional information
?
-
-
EuT substrate specificity, overview, reaction cycle involving EutT and CobA, overview
-
-
-
additional information
?
-
-
the enzyme catalyzes the final step in the conversion of cyanocobalamin, i.e. vitamin B12, to the essential human cofactor adenosylcobalamin, defects in the enzyme through mutations in the gene encoding the enzyme can result in the metabolic disorder known as methylmalonic aciduria, MMA, only two of the three active sites within the trimer contain the bound ATP substrate, twenty residues at the enzymes N-terminus become ordered upon binding of ATP to form an ATP-binding site and an extended cleft that likely binds cobalamin, cobalamin binding site structure involving residue R186, overview
-
-
-
additional information
?
-
Q970Z7, -
indications for cob(I)alamin-binding due to decrease in light absorbance by cob(I)alamin at 388 nm and conversion to a compound with absorbance maximum at 485 nm, different from adenosylcobalamin
-
-
-
additional information
?
-
Q970Z7, -
no ATP: cob(I)alamin adenosyltransferase activity detectable (anaerobic, 20 min, 80C, pH 8, in presence of 1 mM titanium (III) citrate), no increase in light absorbance by presumably adenosylcobalamin at 525 nm
-
-
-
additional information
?
-
-
no in vitro activity by mutants R215K, R225K as well as K78Q, E84K, G87R, D90N, E91K, L92S, S94L, R186W, C189Y, R190C, R191W, E193K, R194G, F212S, S217R, L220P, and L223P, no mediation of base-off transition of adenosylcobalamin (key step of catalytic mechanism) by mutants D64G, F83S, G87R, D90N, E91K, L92S, S94L, C189Y, R191W, E193K, R194G, F212S, S217R, L220P, and L223P
-
-
-
additional information
?
-
-
TTP does not serve as substrate
-
-
-
additional information
?
-
-
ATP:Cobalamin adenosyltransferases catalyze the transfer a 5'-deoxyadenosyl moiety from ATP to cob(I)alamin in the synthesis of the Co-C bond of coenzyme B12
-
-
-
additional information
?
-
-
The PduO-type ATP:corrinoid adenosyltransferase catalyzes the transfer of the adenosyl-group of ATP to Co1+-cobalamin and Co1+-cobinamide substrates to synthesize adenosylcobalamin and adenosylcobinamide, respectively
-
-
-
additional information
?
-
-
MgATP and Cob(II)alamin binding sites, structure comparison overview
-
-
-
additional information
?
-
-
to overcome the thermodynamically challenging Co2+ -> Co1+ reduction, the enzyme drastically weakens the axial ligand-Co2+ bond so as to generate effectively four-coordinate Co2+-corrinoid species, mechanism, overview. The entire hydrophobic pocket below the corrin ring, and not just residue F112, is critical for the removal of the axial ligand from the cobalt center of the Co2+-corrinoids. Large role of the ATP-induced active-site conformational changes with respect to the formation of 4c Co(II)Cbl
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
P29930, -
-
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
P31570
the main role of this enzyme is apparently the conversion of inactive cobalamins to adenosyl cobalamin for 1,2 propanediol degradation
-
-
-
ATP + cob(I)alamin
triphosphate + coenzyme B12
show the reaction diagram
-
involved in vitamin B12 metabolism
-
-
-
ATP + cob(I)alamin
triphosphate + adenosylcobalamin
show the reaction diagram
Q9HIA7
-
-
-
-
ATP + cob(I)alamin
triphosphate + adenosylcobalamin
show the reaction diagram
Q9HIA7, -
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcobalamin
show the reaction diagram
-
polymorphic variants 239K and 239M, biosynthesis of adenosylcobalamin
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
-
adenosyltransferase enzymes lower the thermodynamic barrier of the Co2+ to Co+ reduction needed for the formation of the unique organometalic Co-C bond of adenosylcobalamin
-
?
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 + adenosylcob(III)alamin
show the reaction diagram
-
final step in the conversion of vitamin B12 to coenzyme B12, the latter being required for degradation of 1,2-propanediol
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
last step in the corrinoid adenosylation pathway, EutT and CobA
-
-
ir
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
P31570
last step in the corrinoid adenosylation pathway, EutT and CobA
-
-
ir
ATP + cob(I)alamin
triphosphate + adenosylcob(III)alamin
show the reaction diagram
-
mechanism of adenosylcobalamin biosynthesis
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(I)alamine
show the reaction diagram
-
-
-
-
?
ATP + cob(I)alamin
triphosphate + adenosylcob(I)alamine
show the reaction diagram
-
-
-
-
?
ATP + cobinamide
triphosphate + adenosylcobinamide
show the reaction diagram
-
-
-
-
?
cob(I)alamin + ADP
adenosylcobalamin + diphosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
adenosylation of the corrinoid ring of cob(I)alamin generates coenzyme B12, i.e. adenosylcobalamin or AdoCbl, an essential cofactor used by enzymes that catalyze intramolecular rearrangements, deaminations, dehydrations, reductions, and reductive dehalogenations
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
adenosylcobalamin is a cofactor required by the methylmalonyl-CoA mutase
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
cobalamin assimilation and recycling pathway, overview, enzyme deficiency causes methylmalonic aciduria, MMA, is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000fold greater than normal, overview
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
-
the enzyme is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium
-
-
?
cob(I)alamin + ATP
adenosylcobalamin + triphosphate
show the reaction diagram
Lactobacillus reuteri CRL1098
-
adenosylation of the corrinoid ring of cob(I)alamin generates coenzyme B12, i.e. adenosylcobalamin or AdoCbl, an essential cofactor used by enzymes that catalyze intramolecular rearrangements, deaminations, dehydrations, reductions, and reductive dehalogenations
-
-
?
cob(I)alamin + ATP + H2O + H+
adenosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + CTP + H2O + H+
cytosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + dATP + H2O + H+
deoxyadenosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
-
-
-
?
cob(I)alamin + GTP + H2O + H+
guanosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)alamin + ITP
hypoxanthosylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)alamin + UTP
uranylcobalamin + diphosphate + phosphate
show the reaction diagram
-
low activity
-
-
?
cob(I)inamide + ATP
adenosylcobinamide + triphosphate
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
EutT is necessary and sufficient for growth of a cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source
-
-
-
additional information
?
-
P31570
EutT is necessary and sufficient for growth of a cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source
-
-
-
additional information
?
-
-
the enzyme interacts with the methionine synthase reductase MSR, which catalyzes the reduction of cob(II)almin to cob(I)alamin, both enzymes activate each other, stoichiometry of the MSR-ATR system, overview
-
-
-
additional information
?
-
-
the flavodoxin in vivo reducing agent that serves as the electron donor to the enzyme possesses a reduction potential that is considerably more positive than that of the Co2+/1+ couple of the corrinoid substrate, the enzyme overcomes this challenge by formation of an ATP-activated unique paramagnetic Co2+ corrinoid species by partial conversion, overview
-
-
-
additional information
?
-
-
enzyme defects cause methylmalonic aciduria type B, regulation, overview
-
-
-
additional information
?
-
-
EuT substrate specificity, overview, reaction cycle involving EutT and CobA, overview
-
-
-
additional information
?
-
-
the enzyme catalyzes the final step in the conversion of cyanocobalamin, i.e. vitamin B12, to the essential human cofactor adenosylcobalamin, defects in the enzyme through mutations in the gene encoding the enzyme can result in the metabolic disorder known as methylmalonic aciduria, MMA
-
-
-
additional information
?
-
-
ATP:Cobalamin adenosyltransferases catalyze the transfer a 5'-deoxyadenosyl moiety from ATP to cob(I)alamin in the synthesis of the Co-C bond of coenzyme B12
-
-
-
additional information
?
-
-
The PduO-type ATP:corrinoid adenosyltransferase catalyzes the transfer of the adenosyl-group of ATP to Co1+-cobalamin and Co1+-cobinamide substrates to synthesize adenosylcobalamin and adenosylcobinamide, respectively
-
-
-
COFACTOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
ADP
-
about 80% of the activity with ATP
ATP
-
twenty residues at the enzymes N-terminus become ordered upon binding of ATP to form an ATP-binding site, structure, overview
ATP
-
the beta-phosphate of ATP is required for binding to the enzyme
ATP
-
preferred cofactor, the beta-phosphate is required playing a role in nucleotide recognition and/or binding
ATP
-
ATP binding motif at the protein N terminus
CTP
-
about 40% activity compared to ATP
dATP
-
about 25% of the activity with ATP
flavodoxin A
-
in vitro reduced flavodoxin provides an electron to generate the co(I)rrinoid substrate in the CobA active site, modeling of enzyme ligand interaction, residues R9 and R165 are important for CobA-FldA docking but not to catalysis, overview
-
GTP
-
about 5% activity compared to ATP
UTP
-
about 10% activity compared to ATP
ITP
-
about 8% activity compared to ATP
additional information
-
no activity with AMP as cofactor
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Ca2+
-
activates 2.5fold
Co2+
-
activation, less effective than Mn2+
Co2+
Q9HIA7
activates, KD: 0.27 mM; enzyme depends absolutely on divalent cations in the descending order Mg2+, Mn2+, Co2+, 39% of the activity with Mg2+
Co2+
Q9HIA7
activates, KD: 0.27 mM
Co2+
-
activity with divalent cations in descending order: Mg2+, Mn2+, Co2+
Co2+
-
1.75fold activation
Co2+
-
tetradentate coordination for the cobalt ion, binding structure, overview. In the four-coordinate state, the lower ligand and entire nucleotide arm are displaced by Phe91 and Trp93 and the N-terminal helix from the opposing subunit. This yields a closed active site or conformation for the enzyme. Relative to the five-coordinate cob(II)alamin state, this displacement involves a conformational change in the loop that extends from Met87 to Cys105 and includes a change in the orientation of Phe91 and Trp93
Fe2+
-
required and contained by EutT
K+
-
may be acting upon a step in the reduction of hydroxocobamide
Mg2+
-
activation, less effective than Mn2+
Mg2+
-
activation, less effective than Mn2+
Mg2+
-
activation, less effective than Mn2+
Mg2+
-
-
Mg2+
Q9HIA7
activates, KD: 0.11 mM; enzyme depends absolutely on divalent cations in the descending order Mg2+, Mn2+, Co2+
Mg2+
Q9HIA7
activates, KD: 0.11 mM
Mg2+
-
required for activity, in absence of Mg2+ activity is reduced to 32% of maximum activity, activity with divalent cations in descending order: Mg2+, Mn2+, Co2+
Mg2+
-
activity decreases in absence of Mg2+ by 20%
Mg2+
-
required
Mn2+
Q9HIA7
activates, KD: 0.11 mM; enzyme depends absolutely on divalent cations in the descending order Mg2+, Mn2+, Co2+, 83% of the activity with Mg2+
Mn2+
Q9HIA7
activates, KD: 0.11 mM
Mn2+
-
activity with divalent cations in descending order: Mg2+, Mn2+, Co2+
Ni2+
-
1.5fold activation
titanium citrate
-
-
Zn2+
-
activation, less effective than Mn2+
Zn2+
-
1.25fold activation
Mn2+
-
32% stimulation
additional information
-
no activation by Ca2+ or Cd2+
additional information
-
no activation by Ca2+ or Cd2+
additional information
Q9HIA7
no activity with Ni2+ and Ca2+
additional information
-
Ca2+ has no effect on enzyme activity
additional information
-
no activation by Na+ and Mg2+
additional information
-
no stimulation by Ca2+, Mg2+, Na+, and K+
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
2'-deoxyadenosine
-
weak inhibition
5'-mercapto-5'-deoxyadenosine-5'-triphosphate
-
complete inhibition at a 3 mM concentration
adenine
-
weak inhibition
adenosine
-
weak inhibition
ADP
-
about 90% inhibition
bathophenanthroline
-
inhibition of EutT
Cd2+
-
complete inhibition
cob(I)alamin
-
substrate inhibition at more than 0.008 mM in presence of subsaturating concentrations of ATP (0.003 mM)
cobinamide
-
substrate inhibition at more than 0.005 mM in presence of subsaturating concentrations of ATP (0.003 mM)
cobinamide
-
substrate inhibition
Cu2+
-
complete inhibition
diphosphate
-
28% inhibition
diphosphate
-
about 50% inhibition of EutT
hydrogenobyrinic acid a,c-diamide
-
cobalt-free analogue of cobyrinic acid a,c-diamide
Ni2+
-
complete inhibition
Ni2+
-
62% inhibition, single-site binding
Trimetaphosphate
-
-
-
Triphosphate
-
about 40% inhibition of EutT
TWYYGEAQCDWDD68
-
competitive 30% inhibition of flavodoxin-dependent adenosylcobalamin synthesis by a FldA peptide TWYYGEAQCDWDD68, the inhibition depends on residues Glu61 and Asp68, overview, no inhibition by peptides TWYYGAAQCDWDA68 and WPTAGYHFEASKG132
Zn2+
-
complete inhibition
Zn2+
-
77% inhibition, single-site binding
Mn2+
-
50% inhibition
additional information
-
no inhibition by phosphate
-
additional information
-
no inhibition by cobyrinic acid, hydrogenobyrinic acid
-
additional information
-
alpha,beta-methylene-substituted derivatives of ATP are poor inhibitors
-
additional information
-
CTP, GTP, UTP, and AMP are no inhibitors
-
additional information
-
no inhibition of EutT by phosphate
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
ATP
-
induces formation of activated Co2+ species
coenzyme B12
-
i.e. adenosylcob(III)alamin, is required for stimulation of eut operon expression
coenzyme B12
P31570
i.e. adenosylcob(III)alamin, is required for stimulation of eut operon expression
KCl
Q9HIA7
below 50 mM
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0016
-
2'-Deoxy-ATP
-
-
0.0003
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91Y
0.0012
-
ATP
-
mutant F112Y, Co+ assay; mutant F187A, Co+ assay
0.002
-
ATP
-
mutant F112W, Co+ assay
0.002
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91A/W93D; pH 8.0, 25C, recombinant CobA mutant W93Y
0.0022
-
ATP
-
wild-type, Co+ assay
0.0026
-
ATP
-
mutant F112H, Co+ assay
0.00287
-
ATP
-
mutant D218N, in presence of 0.05 mM cob(I)alamin
0.003
-
ATP
-
pH 8.0, 37C
0.003
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91H; pH 8.0, 25C, recombinant CobA mutant W93A; pH 8.0, 25C, recombinant CobA mutant W93D
0.0031
-
ATP
-
mutant DELTAS183, Co+ assay
0.005
-
ATP
-
mutant T161I, in presence of 0.05 mM cob(I)alamin
0.005
-
ATP
-
wild-type, Co2+ assay
0.00523
-
ATP
-
mutant G97E, in presence of 0.05 mM cob(I)alamin
0.006
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91W/W93F
0.0063
-
ATP
-
recombinant enzyme variant 239K
0.0068
-
ATP
-
pH 8.0, 37C, recombinant wild-type GST-tagged enzyme
0.0069
-
ATP
-
recombinant enzyme variant 239M
0.0069
-
ATP
-
native wild-type, in presence of 0.05 mM cob(I)alamin
0.007
-
ATP
-
pH 8.0, 25C, recombinant wild-type CobA
0.00719
-
ATP
-
N-terminally octa-His tagged wild-type, in presence of 0.05 mM cob(I)alamin
0.00735
-
ATP
-
C-terminally octa-His tagged wild-type, in presence of 0.05 mM cob(I)alamin
0.0079
-
ATP
-
mutant F187A, Co2+ assay
0.00826
-
ATP
-
mutant K78R, in presence of 0.05 mM cob(I)alamin
0.009
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91A; pH 8.0, 25C, recombinant CobA mutant F91W
0.0099
-
ATP
-
mutant DELTAS183, Co2+ assay
0.00997
-
ATP
-
mutant H183Y, in presence of 0.05 mM cob(I)alamin
0.01
-
ATP
-
pH 7.0, 37C, recombinant enzyme
0.0104
-
ATP
-
mutant F112H, Co2+ assay
0.0124
-
ATP
-
mutant G63E, in presence of 0.05 mM cob(I)alamin
0.0141
-
ATP
-
mutant C119Y, in presence of 0.05 mM cob(I)alamin
0.0159
-
ATP
-
mutant F163A, Co+ assay
0.016
-
ATP
-
pH 8.0, 37C
0.0172
-
ATP
-
mutant S68F, in presence of 0.05 mM cob(I)alamin
0.0198
-
ATP
-
pH 8.0, 37C, recombinant full length wild-type enzyme
0.0203
-
ATP
-
mutant G97R, in presence of 0.05 mM cob(I)alamin
0.0205
-
ATP
-
mutant D64G, in presence of 0.05 mM cob(I)alamin
0.026
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant W93H
0.03
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant W93F
0.0346
-
ATP
-
mutant F112A, Co+ assay
0.0459
-
ATP
-
mutant R76G, in presence of 0.05 mM cob(I)alamin
0.073
-
ATP
-
mutant F112W, Co2+ assay
0.0877
-
ATP
-
mutant S126L, in presence of 0.05 mM cob(I)alamin
0.094
-
ATP
-
mutant F112Y, Co2+ assay
0.0961
-
ATP
-
mutant F163A, Co2+ assay
0.11
-
ATP
Q9HIA7
; pH 8.0, 70C, wild-type enzyme, in presence of Mg2+
0.11
-
ATP
Q9HIA7
+/- 0.01
0.215
-
ATP
-
mutant F83S, in presence of 0.05 mM cob(I)alamin
0.32
-
ATP
-
pH 8.0, 37C, recombinant GST-tagged mutant R191W
0.33
-
ATP
Q9HIA7
pH 8.0, 70C, mutant R124A, in presence of Mg2+
1.3
-
ATP
-
pH 8.0, 37C
0.27
-
Co2+
Q9HIA7
pH 8.0, 70C, wild-type enzyme
0.00007
-
cob(I)alamin
-
mutant DELTAS183, Co+ assay
0.00011
-
cob(I)alamin
-
mutant F187A, Co+ assay
0.00013
-
cob(I)alamin
-
-
0.00013
-
cob(I)alamin
-
wild-type, Co+ assay
0.00019
-
cob(I)alamin
-
mutant F163A, Co+ assay
0.00037
-
cob(I)alamin
-
pH 8.0, 37C, recombinant wild-type GST-tagged enzyme
0.00055
-
cob(I)alamin
-
mutant F112Y, Co+ assay
0.00077
-
cob(I)alamin
-
mutant F112W, Co+ assay
0.001
-
cob(I)alamin
-
pH 8.0, 37C
0.001
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91Y
0.0012
-
cob(I)alamin
-
recombinant enzyme variant 239K
0.00158
-
cob(I)alamin
-
N-terminally octa-His tagged wild-type, in presence of 0.5 mM ATP
0.0016
-
cob(I)alamin
-
recombinant enzyme variant 239M
0.0016
-
cob(I)alamin
-
C-terminally octa-His tagged wild-type, in presence of 0.5 mM ATP; native wild-type, in presence of 0.5 mM ATP
0.00172
-
cob(I)alamin
-
mutant D218N, in presence of 0.5 mM ATP
0.00186
-
cob(I)alamin
-
mutant S68F, in presence of 0.5 mM ATP
0.0019
-
cob(I)alamin
-
mutant F112A, Co+ assay
0.00194
-
cob(I)alamin
-
mutant D64G, in presence of 0.5 mM ATP
0.002
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant W93D; pH 8.0, 25C, recombinant CobA mutant W93H
0.00224
-
cob(I)alamin
-
mutant G63E, in presence of 0.5 mM ATP
0.00225
-
cob(I)alamin
-
mutant K78R, in presence of 0.5 mM ATP
0.00252
-
cob(I)alamin
-
mutant G97E, in presence of 0.5 mM ATP
0.00271
-
cob(I)alamin
-
mutant T161I, in presence of 0.5 mM ATP
0.0028
-
cob(I)alamin
-
mutant F112H, Co+ assay
0.003
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91A/W93D; pH 8.0, 25C, recombinant CobA mutant F91H
0.00303
-
cob(I)alamin
-
mutant G97R, in presence of 0.5 mM ATP
0.004
-
cob(I)alamin
-
pH 8.0, 25C, recombinant wild-type CobA
0.0041
-
cob(I)alamin
-
pH 7.0, 37C, recombinant enzyme
0.0045
-
cob(I)alamin
-
pH 8.0, 37C, recombinant full length wild-type enzyme
0.005
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91A; pH 8.0, 25C, recombinant CobA mutant F91W
0.00513
-
cob(I)alamin
-
mutant C119Y, in presence of 0.5 mM ATP
0.00713
-
cob(I)alamin
-
mutant H183Y, in presence of 0.5 mM ATP
0.009
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91W/W93F
0.00909
-
cob(I)alamin
-
mutant R76G, in presence of 0.5 mM ATP
0.011
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant W93A
0.012
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant W93F
0.0143
-
cob(I)alamin
-
mutant S126L, in presence of 0.5 mM ATP
0.016
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant W93Y
0.0302
-
cob(I)alamin
-
mutant F83S, in presence of 0.5 mM ATP
0.06
-
cob(I)alamin
-
pH 8.0, 37C, recombinant GST-tagged mutant R191W
0.000096
-
cob(I)inamide
-
-
0.0078
-
cob(II)alamin
-
wild-type, Co2+ assay
0.015
-
cob(II)alamin
-
mutant F187A, Co2+ assay
0.016
-
cob(II)alamin
-
mutant DELTAS183, Co2+ assay
0.028
-
cob(II)alamin
-
mutant F112W, Co2+ assay
0.034
-
cob(II)alamin
-
mutant F112Y, Co2+ assay
0.053
-
cob(II)alamin
-
mutant F112H, Co2+ assay
0.134
-
cob(II)alamin
-
mutant F163A, Co2+ assay
0.003
-
Cobalamin
Q9HIA7
pH 8.0, 70C, wild-type enzyme, in presence of Mg2+
0.004
-
Cobalamin
Q9HIA7
pH 8.0, 70C, mutant R124A, in presence of Mg2+
0.01
-
cyanocob(I)alamin
-
pH 8.0, 37C, i.e. vitamin B12, reduced form
0.01
-
cyanocob(I)alamin
-
-
0.14
-
dATP
Q9HIA7
; pH 8.0, 70C, wild-type enzyme, in presence of Mg2+
0.14
-
dATP
Q9HIA7
+/- 0.02
0.003
-
hydroxocobalamin
Q9HIA7
-
0.003
-
hydroxocobalamin
Q9HIA7
+/- 0.0004
0.11
-
Mg2+
Q9HIA7
pH 8.0, 70C, wild-type enzyme
0.11
-
Mn2+
Q9HIA7
pH 8.0, 70C, wild-type enzyme
additional information
-
additional information
-
kinetics
-
additional information
-
additional information
-
-
-
additional information
-
additional information
-
-
-
additional information
-
additional information
-
kinetics
-
additional information
-
additional information
-
steady-state kinetics
-
additional information
-
additional information
-
wild-type: KM for CTP and UTP increased relative to ATP; wild-type: KM for GTP and ITP 530-13000fold increased relative to ATP
-
additional information
-
additional information
-
elevated for either ATP or cobalamin or both substrates in case of mutants which show decreased adenosylcobalamin synthesis activity that can be partly corrected by increased hydroxycobalamin concentration; large changes in KM for both substrates for mutants R76G, F83S and S126L; wild-type kinetics by mutants G97E, C119Y, T161I, and H183Y
-
additional information
-
additional information
-
kinetics for wild-type and mutants enzymes from two different assay methods, overview
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.00038
-
ATP
-
mutant F112H, Co+ assay
0.0005
-
ATP
-
mutant F112H, Co2+ assay
0.0016
-
ATP
-
mutant F112Y, Co+ assay
0.0018
-
ATP
-
mutant F112W, Co+ assay
0.0023
-
ATP
-
mutant F112Y, Co2+ assay
0.0028
-
ATP
-
mutant F112W, Co2+ assay
0.0038
-
ATP
-
mutant S129A
0.0056
-
ATP
-
mutant F112A, Co+ assay
0.0062
-
ATP
-
mutant R128K
0.008
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91A/W93D
0.0084
-
ATP
-
mutant R132K
0.01
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91W/W93F; pH 8.0, 25C, recombinant CobA mutant W93F
0.014
-
ATP
-
mutant F187A, Co2+ assay
0.015
-
ATP
-
mutant F163A, Co2+ assay; mutant F163A, Co+ assay
0.017
-
ATP
-
mutant DELTAS183, Co2+ assay
0.02
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91A; pH 8.0, 25C, recombinant CobA mutant F91H
0.021
-
ATP
-
mutant F187A, Co+ assay
0.023
-
ATP
-
mutant DELTAS183, Co+ assay
0.026
-
ATP
-
wild-type, Co+ assay
0.029
-
ATP
-
wild-type, Co2+ assay
0.03
-
ATP
-
pH 7.0, 37C, recombinant enzyme
0.04
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant W93A
0.05
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91Y; pH 8.0, 25C, recombinant CobA mutant W93D
0.07
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant W93H
0.08
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91W; pH 8.0, 25C, recombinant wild-type CobA
0.096
-
ATP
-
mutant D35E/R128K
0.1
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant W93Y
0.118
-
ATP
-
mutant D35N
0.14
-
ATP
Q9HIA7
pH 8.0, 70C, mutant R124A, in presence of Mg2+
0.23
-
ATP
Q9HIA7
; pH 8.0, 70C, wild-type enzyme, in presence of Mg2+
0.23
-
ATP
Q9HIA7
+/- 0.01
0.41
-
ATP
-
mutant D35N/R128K
9
-
ATP
-
mutant D35R/R128D, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin
19.2
-
ATP
-
mutant K78R
29.4
-
ATP
-
mutant G63E
31.8
-
ATP
-
mutant D64G
34.8
-
ATP
-
mutant S126L
43.8
-
ATP
-
mutant D218N
46.8
-
ATP
-
mutant S68F
48
-
ATP
-
mutant G97R
124.2
-
ATP
-
mutant F83S
137.4
-
ATP
-
mutant T161I
154.8
-
ATP
-
mutant R76G
166.2
-
ATP
-
N-terminally octa-His tagged wild-type
169.8
-
ATP
-
mutant G97E; mutant H183Y
243.6
-
ATP
-
mutant C119Y
285
-
ATP
-
native wild-type
0.00041
-
cob(I)alamin
-
mutant R132K
0.00042
-
cob(I)alamin
-
mutant F112H, Co+ assay
0.00065
-
cob(I)alamin
-
mutant D35R/R128D, at subsaturating concentrations of ATP 30 mM ATP and 0.02 mM cob(I)alamin
0.0017
-
cob(I)alamin
-
mutant F112Y, Co+ assay
0.002
-
cob(I)alamin
-
mutant F112W, Co+ assay
0.0024
-
cob(I)alamin
-
mutant D35E/R128K
0.004
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91W/W93F
0.0066
-
cob(I)alamin
-
mutant F112A, Co+ assay
0.0068
-
cob(I)alamin
-
mutant D35N
0.009
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant W93F; pH 8.0, 25C, recombinant CobA mutant W93Y
0.01
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91A/W93D
0.011
-
cob(I)alamin
-
pH 8.0, 37C, recombinant GST-tagged mutant R191W
0.011
-
cob(I)alamin
-
mutant R128K
0.014
-
cob(I)alamin
-
mutant D35N/R128K
0.015
-
cob(I)alamin
-
mutant S129A
0.015
-
cob(I)alamin
-
mutant F163A, Co+ assay
0.018
-
cob(I)alamin
-
mutant F187A, Co+ assay
0.02
-
cob(I)alamin
-
mutant DELTAS183, Co+ assay
0.02
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91A; pH 8.0, 25C, recombinant CobA mutant F91H
0.024
-
cob(I)alamin
-
-
0.024
-
cob(I)alamin
-
wild-type, Co+ assay
0.025
-
cob(I)alamin
-
-
0.037
-
cob(I)alamin
-
pH 8.0, 37C, recombinant wild-type GST-tagged enzyme
0.05
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91Y; pH 8.0, 25C, recombinant CobA mutant W93A; pH 8.0, 25C, recombinant CobA mutant W93D
0.06
-
cob(I)alamin
-
pH 7.0, 37C, recombinant enzyme
0.08
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91W; pH 8.0, 25C, recombinant CobA mutant W93H; pH 8.0, 25C, recombinant wild-type CobA
19.2
-
cob(I)alamin
-
mutant K78R
29.4
-
cob(I)alamin
-
mutant G63E
31.8
-
cob(I)alamin
-
mutant D64G
34.8
-
cob(I)alamin
-
mutant S126L
43.8
-
cob(I)alamin
-
mutant D218N
46.8
-
cob(I)alamin
-
mutant S68F
48
-
cob(I)alamin
-
mutant G97R
124.2
-
cob(I)alamin
-
mutant F83S
137.4
-
cob(I)alamin
-
mutant T161I
154.8
-
cob(I)alamin
-
mutant R76G
166.2
-
cob(I)alamin
-
N-terminally octa-His tagged wild-type
169.8
-
cob(I)alamin
-
mutant G97E; mutant H183Y
243.6
-
cob(I)alamin
-
mutant C119Y
285
-
cob(I)alamin
-
native wild-type
297.6
-
cob(I)alamin
-
C-terminally octa-His tagged wild-type
0.02
-
cob(I)inamide
-
-
0.00067
-
cob(II)alamin
-
mutant F112H, Co2+ assay
0.0027
-
cob(II)alamin
-
mutant F112Y, Co2+ assay
0.0032
-
cob(II)alamin
-
mutant F112W, Co2+ assay
0.015
-
cob(II)alamin
-
mutant F187A, Co2+ assay
0.018
-
cob(II)alamin
-
mutant DELTAS183, Co2+ assay
0.027
-
cob(II)alamin
-
mutant F163A, Co2+ assay
0.038
-
cob(II)alamin
-
wild-type, Co2+ assay
0.11
-
Cobalamin
Q9HIA7
pH 8.0, 70C, mutant R124A, in presence of Mg2+
0.11
-
dATP
Q9HIA7
; pH 8.0, 70C, wild-type enzyme, in presence of Mg2+
0.18
-
hydroxocobalamin
Q9HIA7
-
additional information
-
2'-Deoxy-ATP
-
kcat/KM: 3300 1/M*s
0.0051
-
2'-Deoxy-ATP
-
-
additional information
-
ATP
-
kcat/KM: 12000 1/M*s; mutant D35E/R128K, kcat/KM: 23 1/M*s; mutant D35N, kcat/KM: 52 1/M*s; mutant D35N/R128K, kcat/KM: 32 1/M*s; mutant D35R/R128D, kcat/KM: 0.014 1/M*s, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin; mutant R128K, kcat/KM: 1800 1/M*s; mutant R132K, kcat/KM: 39 1/M*s; mutant S129A, kcat/KM: 4500 1/M*s
additional information
-
ATP
-
C-terminally octa-His tagged wild-type, kcat/KM is 0.67 per microM and min; mutant C119Y, kcat/KM is 0.29 per microM and min; mutant D218N, kcat/KM is 0.25 per microM and min; mutant D64G, kcat/KM is 0.026 per microM and min; mutant F83S, kcat/KM is 0.010 per microM and min; mutant G63E, kcat/KM is 0.039 per microM and min; mutant G97E, kcat/KM is 0.46 per microM and min; mutant G97R, kcat/KM is 0.039 per microM and min; mutant H183Y, kcat/KM is 0.28 per microM and min; mutant K78R, kcat/KM is 0.039 per microM and min; mutant R76G, kcat/KM is 0.056 per microM and min; mutant S126L, kcat/KM is 0.007 per microM and min; mutant S68F, kcat/KM is 0.045 per microM and min; mutant T161I, kcat/KM is 0.46 per microM and min; native wild-type, kcat/KM is 0.69 per microM and min; N-terminally octa-His tagged wild-type, kcat/KM is 0.39 per microM and min
297.6
-
ATP
-
C-terminally octa-His tagged wild-type
additional information
-
cob(I)alamin
-
kcat/KM: 180000 1/M*s; mutant D35E/R128K, kcat/KM: 900 1/M*s; mutant D35N, kcat/KM: 2800 1/M*s; mutant D35N/R128K, kcat/KM: 3800 1/M*s; mutant D35R/R128D, kcat/KM: 5.2 1/M*s, at subsaturating concentrations of 30 mM ATP and 0.02 mM cob(I)alamin; mutant R128K, kcat/KM: 12000 1/M*s; mutant R132K, kcat/KM: 55 1/M*s; mutant S129A, kcat/KM: 65000 1/M*s
additional information
-
cob(I)alamin
-
C-terminally octa-His tagged wild-type, kcat/KM is 3.10 per microM and min; mutant C119Y, kcat/KM is 0.79 per microM and min; mutant D218N, kcat/KM is 0.42 per microM and min; mutant D64G, kcat/KM is 0.27 per microM and min; mutant F83S, kcat/KM is 0.07 per microM and min; mutant G63E, kcat/KM is 0.22 per microM and min; mutant G97E, kcat/KM is 0.94 per microM and min; mutant G97R, kcat/KM is 0.26 per microM and min; mutant H183Y, kcat/KM is 0.40 per microM and min; mutant K78R, kcat/KM is 0.14 per microM and min; mutant R76G, kcat/KM is 0.28 per microM and min; mutant S126L, kcat/KM is 0.04 per microM and min; mutant S68F, kcat/KM is 0.42 per microM and min; mutant T161I, kcat/KM is 0.85 per microM and min; native wild-type, kcat/KM is 2.97 per microM and min; N-terminally octa-His tagged wild-type, kcat/KM is 1.75 per microM and min
0.18
-
Cobalamin
Q9HIA7
pH 8.0, 70C, wild-type enzyme, in presence of Mg2+
additional information
-
CTP
-
kcat/KM: 0.38 1/M*s
0.11
-
dATP
Q9HIA7
+/- 0.01
additional information
-
GTP
-
kcat/KM: 18 1/M*s
0.18
-
hydroxocobalamin
Q9HIA7
+/- 0.01
additional information
-
ITP
-
kcat/KM: 0.65 1/M*s
additional information
-
additional information
-
recombinant truncated enzyme versions
-
additional information
-
additional information
-
wild-type, decreased kcat for CTP and UTP relative to ATP; wild-type, kcat for GTP and ITP is not decreased relative to ATP
-
additional information
-
additional information
-
kcat of N-terminal octa-His tagged enzyme is 58% of native or C-terminal tagged enzyme possibly due to interference of N-terminal tag with base-off transition of adenosylcobalamin by the enzyme
-
additional information
-
additional information
-
adenosylation of cobalamin and cobinamide (lacking dimethylbenzimidazole moiety) at similar rates
-
additional information
-
UTP
-
kcat/KM: 0.048 1/M*s
kcat/KM VALUE [1/mMs-1]
kcat/KM VALUE [1/mMs-1] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.057
-
ATP
-
mutant F112H, Co2+ assay
22040
0.14
-
ATP
-
mutant F112H, Co+ assay
22040
0.16
-
ATP
-
mutant F163A, Co2+ assay
22040
0.17
-
ATP
-
mutant F112A, Co+ assay
22040
0.25
-
ATP
-
mutant F112Y, Co2+ assay
22040
0.3
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant W93F
22040
0.39
-
ATP
-
mutant F112W, Co2+ assay
22040
0.9
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91W
22040
0.98
-
ATP
-
mutant F112W, Co+ assay
22040
1.4
-
ATP
-
mutant F112Y, Co+ assay
22040
1.7
-
ATP
-
mutant DELTAS183, Co2+ assay
22040
1.8
-
ATP
-
mutant F163A, Co+ assay; mutant F187A, Co2+ assay
22040
2
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91A; pH 8.0, 25C, recombinant CobA mutant F91W/W93F
22040
3
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant W93H
22040
5
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91A/W93D
22040
5.5
-
ATP
-
wild-type, Co2+ assay
22040
6
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91H
22040
7
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant W93Y
22040
7.5
-
ATP
-
mutant DELTAS183, Co+ assay
22040
10
-
ATP
-
pH 8.0, 25C, recombinant wild-type CobA
22040
12
-
ATP
-
wild-type, Co+ assay
22040
17
-
ATP
-
mutant F187A, Co+ assay
22040
20
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant W93A; pH 8.0, 25C, recombinant CobA mutant W93D
22040
200
-
ATP
-
pH 8.0, 25C, recombinant CobA mutant F91Y
22040
0.4
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91W/W93F
20479
0.6
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant W93Y
20479
0.8
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant W93F
20479
2.6
-
cob(I)alamin
-
mutant F112W, Co+ assay
20479
3.1
-
cob(I)alamin
-
mutant F112Y, Co+ assay
20479
3.6
-
cob(I)alamin
-
mutant F112A, Co+ assay
20479
4
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91A; pH 8.0, 25C, recombinant CobA mutant W93A
20479
5
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91A/W93D; pH 8.0, 25C, recombinant CobA mutant F91H
20479
15
-
cob(I)alamin
-
mutant F112H, Co+ assay
20479
20
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91W; pH 8.0, 25C, recombinant CobA mutant W93D; pH 8.0, 25C, recombinant wild-type CobA
20479
40
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant W93H
20479
50
-
cob(I)alamin
-
pH 8.0, 25C, recombinant CobA mutant F91Y
20479
79
-
cob(I)alamin
-
mutant F163A, Co+ assay
20479
160
-
cob(I)alamin
-
mutant F187A, Co+ assay
20479
180
-
cob(I)alamin
-
-
20479
180
-
cob(I)alamin
-
wild-type, Co+ assay
20479
395
-
cob(I)alamin
-
mutant DELTAS183, Co+ assay
20479
210
-
cob(I)inamide
-
-
144757
0.013
-
cob(II)alamin
-
mutant F112H, Co2+ assay
8738
0.083
-
cob(II)alamin
-
mutant F112Y, Co2+ assay
8738
0.11
-
cob(II)alamin
-
mutant F112W, Co2+ assay
8738
0.2
-
cob(II)alamin
-
mutant F163A, Co2+ assay
8738
0.93
-
cob(II)alamin
-
mutant F187A, Co2+ assay
8738
1.1
-
cob(II)alamin
-
mutant DELTAS183, Co2+ assay
8738
4.8
-
cob(II)alamin
-
wild-type, Co2+ assay
8738
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0013
-
TWYYGEAQCDWDD68
-
37C, recombinant wild-type enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.000125
-
-
activity measured using washed ribosomes; pH 8.0, 37C
0.0005
-
-
in presence of 1 mM ATP, 20 mM NADH, 2 mM FMN, 0.5 mM hydroxycobalamin and NAD(P)H: flavin oxidoreductase, 1.5 mM MgCl2, 100 mM KCl, pH 6
0.001
-
-
purified enzyme, pH 8.0
0.0061
-
-
strain carrying the complementation plasmid eutT+, in vivo activity
0.0061
-
P31570
strain carrying the complementation plasmd eutT+, in vivo activity
0.02
-
-
purified recombinant enzyme
0.023
-
-
reductant: PduS protein, specific activity of LrPduO using different electron sources for the adenosylation of cob(II)alamin
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
0.034
-
Q9HIA7
recombinant mutant R124F
0.045
-
-
reductant: NADPH-dependent ferredoxin protein reductase, specific activity of LrPduO using different electron sources for the adenosylation of cob(II)alamin
0.047
-
-
bovine ATR containing mitochondrial targeting sequence, expressed in E. coli, soluble fraction; pH 8.0, 37C
0.053
-
-
activity at final purification step; pH 8.0, 37C
0.061
-
-
human ATR containing mitochondrial targeting sequence, expressed in E. coli and forming inclusion bodies; pH 8.0, 37C
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
-
-
in the presence of cobalamin as substrate; pH 8.0, 30C
0.089
-
-
purified recombinant GST-tagged wild-type enzyme
0.091
-
-
purified recombinant full length wild-type enzyme, in absence of Mg2+
0.098
-
-
human ATR with no mitochondrial targeting sequence, expressed in E. coli and forming inclusion bodies; pH 8.0, 37C
0.13
-
-
in the presence of cobyrinic acid a,c-diamide as substrate; pH 8.0, 30C
0.14
-
-
in the presence of cobyric acid as substrate; pH 8.0, 30C
0.14
-
-
reductant: dihydroflavin, specific activity of LrPduO using different electron sources for the adenosylation of cob(II)alamin
0.143
-
-
purified recombinant wild-type enzyme
0.18
-
-
in the presence of cobinamide as substrate; pH 8.0, 30C
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
0.25
-
-
reductant: Ti(III)citrate, specific activity of LrPduO using different electron sources for the adenosylation of cob(II)alamin
0.262
-
-
purified recombinant full length wild-type enzyme, in presence of 2.5 mM MgCl2
0.28
-
Q9HIA7
dATP
0.28
-
Q9HIA7
+/- 0.01, dATP
0.312
-
P31570
activity of inclusion bodies formed with enzyme expressed in E.coli; pH 8.0, 37C
0.385
-
Q9HIA7
recombinant mutant R124A
0.47
-
Q9HIA7
hydroxocobalamin
0.47
-
Q9HIA7
+/- 0.02, hydroxocobalamin
0.49
-
-
reductant: Ti(III)citrate, specific activity of hATR using different electron sources for the adenosylation of cob(II)alamin
0.61
-
Q9HIA7
ATP
0.61
-
Q9HIA7
+/- 0.02, ATP
0.619
-
Q9HIA7
recombinant wild-type enzyme
additional information
-
-
data for ATR inclusion bodies from the enzyme expressed in E. coli with no mitochondrial targeting sequence also available
additional information
-
-
activities of recombinant truncated enzyme versions
additional information
-
-
activity of mutant enzymes, overview
additional information
-
-
nucleotide substrate specificty
additional information
-
-
substantially reduced Vmax for mutants G63E, D64G, S68F, K78R, G97R and D218N; wild-type kinetics by mutants G97E, C119Y, T161I, and H183Y
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7
-
-
assay at
8
-
-
phosphate or Tris buffer
8
-
-
assay at
8
-
-
assay at
8
-
Q9HIA7
; assay at
8
-
-
assay at
8
-
-
assay at
8
-
-
assay at
8
-
-
assay at
8
-
Q970Z7, -
assay at
8
-
-
assay at
8
-
-
assay at
8
-
-
assay at
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7
8.5
-
30% of maximal activity at pH 7.5
7.5
8.5
-
75% of maximal activity at pH 7.5, 94% at pH 8.5
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
25
-
-
assay at
37
-
-
assay at
37
-
-
assay at
37
-
-
assay at
37
-
-
assay at
37
-
-
assay at
37
-
-
assay at
37
-
-
assay at
70
-
Q9HIA7
; assay at
80
-
Q970Z7, -
assay at
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
-
mRNA expression detected
Manually annotated by BRENDA team
-
skin fibroblasts show low abundance
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
enzyme contains a putative mitochondrial targeting sequence
Manually annotated by BRENDA team
-
Arg19 in the mitochondrial targeting sequence is important
Manually annotated by BRENDA team
-
from skin cells
-
Manually annotated by BRENDA team
Sulfolobus tokodaii 7
-
-
-
-
Manually annotated by BRENDA team
additional information
-
subcellular distribution
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
Bacillus cereus (strain ATCC 14579 / DSM 31)
Bacillus cereus (strain ATCC 14579 / DSM 31)
Bacillus subtilis (strain 168)
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Sulfolobus tokodaii (strain DSM 16993 / JCM 10545 / NBRC 100140 / 7)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
17890
-
Q970Z7, -
MALDI-TOF analyses of recombinant hexa-His tagged protein, N-terminal deletion due to cleavage between Thr12 and Lys13, proportion of this species increases over time
19200
-
Q970Z7, -
theoretical, deduced from primary sequence
19230
-
Q970Z7, -
MALDI-TOF analyses of recombinant hexa-His tagged protein, N-terminal deletion due to cleavage between Thr11 and Asn12; matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy
20400
-
Q970Z7, -
theoretical, deduced from primary sequence
36600
-
P31570
calculated from amino acid sequence
37000
-
P31570
SDS-PAGE of recombinant enzyme expressed in E. coli
44000
-
-
gel filtration
52000
-
-
SDS-PAGE, recombinant enzyme without mitochondrial targeting sequence
54000
-
-
calculated from amino acid sequence of recombinant enzyme without mitochondrial targeting sequence
55000
-
-
calculated from amino acid sequence of recombinant enzyme without mitochondrial targeting sequence
56000
-
-
HPLC gel filtration
56000
-
-
SDS-PAGE, recombinant enzyme with mitochondrial targeting sequence
58000
-
-
calculated from amino acid sequence of recombinant enzyme with mitochondrial targeting sequence
60000
-
Q970Z7, -
extra band of recombinant hexa-His tagged protein in Western blotting
additional information
-
Q970Z7, -
no extra band of recombinant hexa-His tagged protein in Western blotting
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 36812, sequence calculation, full length enzyme
?
Q970Z7, -
x * 19230, recombinant enzyme with a 6His-tag, matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy. The 6His-tagged recombinant enzyme can exist as an oligomer in SDS-PAGE
?
Sulfolobus tokodaii 7
-
x * 19230, recombinant enzyme with a 6His-tag, matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy. The 6His-tagged recombinant enzyme can exist as an oligomer in SDS-PAGE
-
dimer
-
2 * 28000, SDS-PAGE
trimer
Q9HIA7
; gel-filtration
trimer
Q9HIA7
gel filtration
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
trimer
Q970Z7, -
3 * 20000, revealed by size-exclusion chromatography and crystal structure analysis
trimer
-
wild-type and mutants D35N and R128K, revealed by gel filtration chromatography
trimer
-, Q2SZ09
three molecules in asymmteric unit, oligomerisation mediated by ionic interactions and hydrogen bondings
additional information
Q9HIA7
each subunit is composed of a bundle of 5 alpha-helices, the active site lies at the junction between the subunits
additional information
-
approximate molecular weight of full length and truncated mutants
additional information
-
structural comparisons between apo BcPduO and BcPduO in complex with MgATP reveal that the N-terminal strands of both structures are ordered, which is in contrast with the most previously available PduO-type adenosyltransferase structures. Apo BcPduO is bound to additional dioxane molecules causing a side chain conformational change at the Tyr30 residue, which is an important residue that mediates hydrogen bonding with ATP molecules upon binding of cobalamin to the active site
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
apoenzyme and in complex with Mg2+/ATP, C-centred orthorhombic space group C222(1), unit cell parameters: a: 64.93 A, b: 137.08 A, c: 158.55 A, alpha, beta, gamma: 90, one trimer in the asymmetric unit, sitting-drop combined with hanging-drop vapour-diffusion method: 13 mg/ml protein solution, precipitants: 1.55-1.6 M ammonium sulfate, 9-10% (v/v) dioxane (pH 6.5), for complex: 4 mM ATP and 4 mM Mg2+
-
purified recombinant PduO in complex with ATP, hanging drop vapor diffusion method, mixing o 0.001 ml of 13 mg/ml protein solution, with or without 4 mM ATP and 4 mM MgCl2, with 0.001 ml of optimized reservoir solution containing 100 mM MES, pH 6.5, 1.52 M ammonium sulfate, 9% v/v dioxane, followed by equilibration over 0.5 ml of the mother liquor, the cryoprotection solution contains 50 mM MES, pH 6.5, 0.76 M ammonium sulfate, 4.5% v/v dioxane, and 1.7 M sodium malonate, pH 7.0, X-ray diffraction structure determination and analysis
-
native (PDB: 2ZHY, with ordered N-terminal loop and formed active site) and in complex with ATP (PDB: 2ZHZ, substrate-binding cleft is widened and the N-terminal loop swung out and conformational shift of Arg129 side chain upon ATP-binding, similar structure to human and Lactobacillus reuteri PduO except for interaction of NH2 nitrogen atom of Arg11 with gamma-phosphate of ATP), five alpha helix bundle, monomeric subunits almost identical conformations in both structures, crystals: orthothrombic space group C222(1), three monomers in the asymmetric unit, unit cell parameters: a: 52.55/53.91, b: 148.98/148.17, c: 157.35/158.10, microcrystals (from sitting-drop vapour-diffusion at 22C) scaled up by hanging-drop vapour diffusion, reservoir solution (pH5.7, 22% (w/v) isopropanol, 12% (v/v) PEG4000), PduO-MgATP complex: ATP soaked into native PduO crystals, molecular replacement using PDB: 2G2D as model
-, Q2SZ09
mutants D35N (without tag) in complex with ATP and cob(II)alamin and R132K (without tag) in complex with ATP, thin plate crystals, space group P6(3), two monomers in the asymmetric unit, unit cell parameters: a, b: 65A, c: 169A, beta: 90; vapour-diffusion under anoxic conditions, protein solution (15 mg/ml, containing ATP, hydroxycobalamin and a reducing system of NADH, FMN, and flavodoxin reductase), reservoir solution (incl. 14-16% PEG8000, pH6)
-
purified recombinant His-tagged enzyme in complex with its substrates, hanging drop vapour diffusion method, 20C, 0.004 ml of 20 mg/ml protein in 10 mM Tris-HCl, pH 8.0, is mixed with 0.004 ml od precipitant solution containing 0.1 M HEPES, pH 8.5, 1.1 M ammonium sulfate, 2 mM ATP, 55 mM MgCl2, 165 mM NaCl, and 15 mM cob(I)alamin, 7 days, X-ray diffraction structure determination and analysis at 1.68 A resolution
-
trimer of three independent five-helix bundles, active sites at the interface between adjacent monomers, no significant structural changes accompany catalysis, precatalytic complex with ATP: cob(II)alamin (PDB: 3CI1, four-coordinate, base-off cob(II)alamin intermediate, enzyme with fully ordered six C-terminal residues and potassium ion in active site), complex with tripolyphosphate: adenosylcobalamin (PDB: 3CI3, partially occupied with five-coordinate adenosylcobalamin), precatalytic complex with ATP: cob(II)inamide (PDB: 3CI4, cob(II)inamide-binding structurally indistinguishable from cob(II)alamin-binding), binding of cobalamin and cobinamide (lacking dimethylbenzimidazole moiety) in identical positions and orientation, space group R3, one molecule in asymmetric unit, unit cell parameters: a: 67.8-68, b: 67.8-68, c: 110.9-111.3, beta: 90, molecular replacement using PDB: 2NT8 as model; vapour-diffusion with tag-cleaved protein solution (18-22 mg/ml, in presence of hydroxycobalamin and/or adenosylcobalamin or dicyanocobinamide, ATP etc.) and reservoir solution (10-13% (w/v) PEG 8000, pH 6), cubic crystals, crystallisation under anoxic conditions in presence of flavin-dependent reducing system
-
purified recombinant CobA in complex with ATP, four-coordinate cobalamin, and five-coordinate cobalamin, hanging drop vapour diffusion method, 0.002 ml of 10 mg/ml CobA protein in 20 mM Tris-HCl, pH 8.0, 20 mM NADH, 3 mM ATP, 4.5 mM MgCl2, and 2 mM HOCbl, is mixed with 0.002 ml of well solution containing 100 mM MES, pH 6.0, 320 mM NaCl, and 19.6% w/v PEG4000, X-ray diffraction structure determination and analysis at 1.95 A resolution
-
3fold symmetric trimer of five counterclockwise helix bundles, one molecule in asymmetric unit, polypropylene glycol 400 molecule captured in putative active site (positively charged, important residues: Asp32, Arg118), crystals of selenomethionine derivative: space group P2(1)3, unit cell parameters: a, b, c: 84.67 A, hanging-drop vapour-diffusion method: protein solution (15 mg/ml) + reservoir solution (pH 8.1, 2.5 M ammonium sulphate, 2% polypropylene glycol 400); hanging-drop vapor diffusion method; sparse matrix method at 20C; trimer with noncrystallographic 3fold symmetry in the asymmetric unit, consistent of five helix bundles, identical topology to ST1454 but less ion pairs around the putative active site, crystals: space group P4(1)2(1)2, unit cell parameters: a, b: 117.58 A, c: 79.05 A, hanging-drop vapour-diffusion method: protein solution (21 mg/ml) + reservoir solution (pH6.8, 0.5 M ammonium sulfate, 2% polypropylene glycol 400), molecular replacement
Q970Z7, -
purified recombinant wild-type and selenomethionine-labeled enzymes, crystal growth from 0.4 M ammonium phosphate, 4% methyl-pentanediol, 5% glycerol, at 20C, X-ray diffraction structure determination and analysis at 1.5-1.9 A resolution
Q9HIA7
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4
-
-
stable for several weeks with dithiothreitol
55
-
-
sharp decrease in activity with preincubation at 55C or higher
60
-
-
no appreciable loss of activity after 10 min
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
2-mercaptoethanol stabilizes during purification
-
repeated freeze-thawing inactivates
-
2-mercaptoethanol stabilizes during purification
-
repeated freeze-thawing inactivates
-
DTT stabilizes during purification and storage, removal leads to complete loss of activity
-
dithiothreitol stabilizes the enzyme
-
secondary structure is retained even in 7 M guanidine hydrochroride
Q970Z7, -
secondary structure stable even at 7 M guanidine hydrochloride as revealed by circular dichroism spectrophotometry, stability may be due to high numbers of ion pairs and ion pair networks around the active site (especially Glu125) and 3fold axis
Q970Z7, -
secondary structure starts melting in presence of 3.5 M guanidine hydrochloride and is completely denatured at 4.5 M as revealed by circular dichroism spectrophotometry, possibly due to lower numbers of ion pairs and ion pair networks around the putative active site and 3fold axis compared to homolog ST1454
Q970Z7, -
2-mercaptoethanol stabilizes the enzyme during purfication
Q9HIA7
ORGANIC SOLVENT
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
guanidine-HCl
Q970Z7, -
7 M, activity is retained
guanidine-HCl
Sulfolobus tokodaii 7
-
7 M, activity is retained
-
OXIDATION STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
enzyme is oxygen-labile
-
658993
EutT is oxygen-labile, exposure to air leads to complete loss of activity
-
659048
EutT is oxygen-labile, exposure to air leads to complete loss of activity
P31570
659048
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
-20C, lyophilized, at least 1 week
-
2-mercaptoethanol stabilizes during storage
-
4C, about 1 month
-
-20C, more than 50% loss of activity within 1 week, with 50% glycerol only 20% loss of activity
-
2-mercaptoethanol stabilizes during storage
-
DTT stabilizes during storage
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
nickel-chelating HiTrap chromatography (elution with 1 M imidazole), HiLoad 16/60 Superdex 200 gel filtration chromatography (2 mM dithiothreitol), concentration to 13 mg/ml by ultracentrifugation
-
recombinant PduO from Escherichia coli strain BL21 (DE3) cell culture medium
-
from crude lysate by HiTrap Ni2+-chelating affinity chromatography (gradient elution), gel filtration on HiLoad 16/60 Superdex 200 (pH7.9, 4 mM dihiothreitol), concentration to 15 mg/ml by ultrafiltration, yield: 45 mg pure protein per litre culture
-, Q2SZ09
protamine sulfate, heat treatment and chromatography on Sephadex G-25 and DEAE-cellulose
-
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
-
after expression at 16C, lysis with French pressure cell, pH 7.5, and centrifugation
-
centrifugation and SDS-PAGE
-
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
-
partial purification of the enzyme is achieved from ribosomal fraction by salt extraction and ammonium sulfate fractionation, followed by two chromatographic steps
-
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)
-
recombinant His-tagged wild-type and mutant PduOs from Escherichia coli by nickel affinity chromatography to over 99% homogeneity
-
using a HisTrap FF column
-
using Ni-NTA chromatography
-
native enzyme by anion exchange chromatography, dialysis, and hydrophobic interaction chromatography, recombinant enzyme from Escherichia coli strain BL21(DE3)
-
centrifugation, Mono Q chromatography and gel filtration
-
recombinant His-tagged EutT from Escherichia coli to 70% purity
-
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)
-
recombinant solubilized enzyme from Escherichia coli 1.4fold to homogeneity, partial purification of recombinant truncated mutants, recombinant N-terminal domain to homogeneity
-
recombinant tagless, wild-type CobA, and His7-tagged mutant enzymes by nickel affinity chromatography, the His-tag is cleaved off by rTEV protease
-
ammonium sulfate precipitation followed by two chromatographic steps
-
; from bacterial lysate by heat treatment (30 min, 70C) and chromatography: HiTrap Phenyl FF column (2-0 M NaCl gradient, pH 8) and HiLoad 26/60 Superdex 75-pg column (pH 8), for the selenomethionine derivative: HiTrap QXL column (0-2 M NaCl gradient, pH 9) and HiLoad 26/60 Superdex 75-pg column (pH 8); from bacterial lysate by heat treatment (30 min, 70C) and chromatography: HiTrap QXL column (0-2 M NaCl gradient, pH9) and HiLoad 26/60 Superdex 75-pg column (pH8)
Q970Z7, -
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
from genomic DNA in pET-28a for expression in Escherichia coli BL21 (DE3)
-
gene pduO, overexpression in Escherichia coli strain BL21 (DE3) and secretion to the medium
-
expressed in Escherichia coli
-
from genomic DNA in pET28a for expression with a C-terminal hexa-His tag in Escherichia coli BL21 (DE3)
-, Q2SZ09
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
-
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
-
expressed in Escherichia coli as an N-terminal His-tagged fusion protein
-
gene pduO, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
-
gene pduO, recombinant overexpression of His-tagged wild-type and mutant PduOs in Escherichia coli
-
QuickChangeXL site-directed mutagenesis performed on wild-type LrpduO in pTEV3 before expression of wild-type and variants in Escherichia coli BL21(DE3) with a rTEV protease-cleavable N-terminal hexa-His tag
-
gene cobAMm, expression in a Salmonella enterica strain lacking the housekeeping CobA enzyme restores coenzyme B12 synthesis, overexpression in Escherichia coli strain BL21(DE3)
-
recombinantly expressed in Escherichia coli
-
structural gene cobO
-
2 enzymes encoded by genes eutT and cobA, DNA sequence determination, analysis of several geno- and phenotypic strains, enzyme is expressed e.g. as fusion protein or to complement a deficient strain, overview
-
expression of wild-type enzyme in Escherichia coli in inclusion bodies, expression of truncated mutants in Escherichia coli
-
gene cobA, recombinant overexpression of tagless, wild-type CobA and expression of His7-tagged mutant CobAs in Escherichia coli strain BL21(lambdaDE3) carrying a null allele of btuR, the cobA homologue in this bacterium
-
gene eutT, overexpression of His-tagged enzyme in Escherichia coli as insoluble protein
-
overexpression of wild-type and mutant enzymes in Escherichia coli using strain BL21(DE3) for the His-tagged variant, and several derivatives of strain DH5alpha, overview
-
structural gene cobA
-
2 enzymes encoded by genes eutT and cobA, DNA sequence determination, analysis of several geno- and phenotypic strains, enzyme is expressed e.g. as fusion protein or to complement a deficient strain, overview
P31570
expression in Escherichia coli as a 6His-tagged fusion protein; expression in Escherichia coli as a His-tagged protein; from genomic DNA in pET20b for expression with N-terminal hexa-His tag in Escherichia coli strain BL21 (DE3); from genomic DNA in pET20b for expression with N-terminal hexa-His tag in Escherichia coli strain BL21 (DE3) or B834 (DE3) for the selenomethionine derivative
Q970Z7, -
; expression in Escherichia coli in strain BL21(DE3) of the wild-type enzyme and in strain B834(DE3) as selenomethionine-labeled enzyme
Q9HIA7
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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
-
site-directed mutagenesis, the mutant enzyme is not expressed
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)
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
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
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
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
-
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/A135T
-
site-directed mutagenesis, the mutant enzyme shows 30% reduced activity compared to the wild-type enzyme
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
D35E/R128K
-
reduced activity to lower extent than mutation R128K alone
D35N
-
230fold decrease in kcat/KM (ATP) most likely due to disruption of salt bridge with residue R128 as observed in crystal structure
D35N
-
site-directed mutagenesis
D35N/R128K
-
similar kinetics as mutation D35N alone
D35R/R128D
-
reciprocal mutation to D35/R128, very high KM values did not allow for kinetic analyses at saturating substrate concentrations
DELTAS183
-
Km (mM) (Co+ assay): 0.0031 (ATP), 0.00007 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.023 (ATP), 0.02 (cob(I)alamin), Km (mM) (Co2+ assay): 0.0099 (ATP), 0.0163 (cob(II)alamin), kcat (1/sec) (Co+ assay): 0.017 (ATP), 0.018 (cob(II)alamin)
F112A
-
Km (mM) (Co+ assay): 0.0346 (ATP), 0.0019 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.0056 (ATP), 0.006 (cob(I)alamin). Mutant F112A shows no activity in the Co2+ assay. The crystal structure of mutant F112A reveal that cob(II)alamin binds to the active site as a five-coordinate species with 5,6-dimethylbenzimidazole serving as a fifth axial ligand. In the Co+ assay [adenosylation of cob(I)alamin], mutant F112A is catalytically competent and displays only a slight decrease in kcat, supposing that in the absence of a four-coordinate cob(II)alamin species, the enzyme is inactive
F112A
-
mutation of the conserved Phe-112 in the active site of PduO is critical for the displacement of cob(II)alamin and leads to an inactive enzyme
F112A
-
site-directed mutagenesis
F112H
-
Km (mM) (Co+ assay): 0.0026 (ATP), 0.0028 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.00038 (ATP), 0.0042 (cob(I)alamin), Km (mM) (Co2+ assay): 0.01 (ATP), 0.053 (cob(II)alamin), kcat (1/sec) (Co+ assay): 0.00059 (ATP), 0.00067 (cob(II)alamin). The crystal structure of an mutantF112H variant show a 5,6-dimethylbenzimidazole-off/His-on interaction between the corrinoid and the enzyme, whose catalytic efficiency is 4 orders of magnitude lower than that of the wild-type protein. The analysis of the kinetic parameters of mutantF112H suggests that the F112H substitution negatively impacts product release
F112H
-
site-directed mutagenesis
F112W
-
Km (mM) (Co+ assay): 0.002 (ATP), 0.00077 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.0018 (ATP), 0.002 (cob(I)alamin), Km (mM) (Co2+ assay): 0.0073 (ATP), 0.028 (cob(II)alamin), kcat (1/sec) (Co+ assay): 0.0028 (ATP), 0.0032 (cob(II)alamin)
F112Y
-
Km (mM) (Co+ assay): 0.0012 (ATP), 0.00055 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.0016 (ATP), 0.0017 (cob(I)alamin), Km (mM) (Co2+ assay): 0.0094 (ATP), 0.034 (cob(II)alamin), kcat (1/sec) (Co+ assay): 0.0023 (ATP), 0.0027 (cob(II)alamin)
F163A
-
Km (mM) (Co+ assay): 0.0159 (ATP), 0.00019 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.015 (ATP), 0.015 (cob(I)alamin), Km (mM) (Co2+ assay): 0.096 (ATP), 0.134 (cob(II)alamin), kcat (1/sec) (Co+ assay): 0.015 (ATP), 0.027 (cob(II)alamin)
F187A
-
Km (mM) (Co+ assay): 0.0012 (ATP), 0.00011 (cob(I)alamin), kcat (1/sec) (Co+ assay): 0.021 (ATP), 0.018 (cob(I)alamin), Km (mM) (Co2+ assay): 0.0079 (ATP), 0.0158 (cob(II)alamin), kcat (1/sec) (Co+ assay): 0.014 (ATP), 0.015 (cob(II)alamin)
F187A
-
site-directed mutagenesis
R128K
-
kinetics similar to wild-type, R128 conserved among PduO-type ACAs, R128 and R128K build salt bridge to residue D35 of adjacent unit, mutation R128W most common in methylmalonic aciduria patients
R128K
-
site-directed mutagenesis
R132K
-
60-80fold decrease in kcat with respect to both substrates, 300fold decrease in kcat/KM (ATP), 3000fold decrease in kcat/KM (cob(I)alamin), identical position of ATP in the crystal structure compared to wild-type, no cob(I)alamin detectable in crystal structure
R132K
-
site-directed mutagenesis
S129A
-
mildly affected kcat and KM for both substrated
S159A
-
site-directed mutagenesis
A134L
-
site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme
C79A
-
site-directed mutagenesis of EutT, the mutant shows highly reduced activity compared to the wild-type enzyme
C80A
-
site-directed mutagenesis of EutT, the mutant shows 99% reduced activity compared to the wild-type enzyme
C83A
-
site-directed mutagenesis of EutT, the mutant shows 99% reduced activity compared to the wild-type enzyme
F91A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F91A,W93A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F91D
-
site-directed mutagenesis, the mutant is inactive
F91H
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
F91W
-
site-directed mutagenesis, the mutant shows the same activity as the wild-type enzyme
F91W,W93F
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
R165A
-
site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme
R165E
-
site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme
R98A
-
site-directed mutagenesis, the mutant shows 80% reduced activity compared to the wild-type enzyme
R98E
-
site-directed mutagenesis, the mutant shows about 30% reduced activity compared to the wild-type enzyme
R98E/R165E
-
site-directed mutagenesis, the mutant shows 85% reduced activity compared to the wild-type enzyme
R9A
-
site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme
R9E
-
site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme
R9E/R165E
-
site-directed mutagenesis, the mutant shows 30% reduced activity compared to the wild-type enzyme
R9E/R98E
-
site-directed mutagenesis, the mutant shows 60% reduced activity compared to the wild-type enzyme
R9E/R98E/R165E
-
site-directed mutagenesis, the mutant shows 25% reduced activity compared to the wild-type enzyme
W93A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W93D
-
site-directed mutagenesis, the mutant shows the same activity as the wild-type enzyme
W93F
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W93H
-
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
W93Y
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E126A
Q9HIA7
no catalytic activity; site-directed mutagenesis, inactive mutant
E126A
Q9HIA7
inactive mutant
E126K
Q9HIA7
no catalytic activity; site-directed mutagenesis, inactive mutant
E126K
Q9HIA7
inactive mutant
R119A
Q9HIA7
no catalytic activity; site-directed mutagenesis, inactive mutant
R119A
Q9HIA7
inactive mutant
R119W
Q9HIA7
no catalytic activity; site-directed mutagenesis, inactive mutant
R119W
Q9HIA7
inactive mutant
R124A
Q9HIA7
about half as active as wild-type; site-directed mutagenesis, about 40% reduced activity compared to the wild-type enzyme
R124A
Q9HIA7
reduced activity
R124F
Q9HIA7
decreased kcat and increased KM; site-directed mutagenesis, about 95% reduced activity compared to the wild-type enzyme
R124F
Q9HIA7
reduced activity
R124K
Q9HIA7
no catalytic activity; site-directed mutagenesis, inactive mutant
R124K
Q9HIA7
inactive mutant
R124W
Q9HIA7
no catalytic activity; site-directed mutagenesis, inactive mutant
R124W
Q9HIA7
inactive mutant
L92S
-
inactive in vitro (10fold excess of substrate compared to standard)
additional information
-
enzyme mutations at residues Gly97, Ser174, Arg186, Arg190, Arg191, Glu193, and Gln234 can result in the metabolic disorder known as methylmalonic aciduria, MMA, overview
additional information
-
construction of transgenic C57/Bl6 mice by expression of the human enzyme using an adeno-associated virus vector with primer pairs specific to the cytomegalovirus enhancer/chicken beta-actin, CBAT, promoter, quantitative and semiquantitative expression analysis in murine liver, overview
V186A
-
site-directed mutagenesis
additional information
-
enzyme expression restores coenzyme B12 synthesis in a Salmonella enterica strain lacking the housekeeping CobA enzyme
F91Y
-
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
additional information
-
construction of several truncated mutants, the activity decreases with truncation degree, remaining residues 1-90 are inactive, overview
additional information
-
truncation of the 9-amino acid N-terminal helix of CobA reduces its FldA-dependent cobalamin adenosyltransferase activity by 97.4%, but shows 4fold higher specific activity than the wild-type enzyme when cob(I)alamin is generated chemically in situ, e.g. by use of KBH4, residues Arg9 and Arg165 of CobA are critical for FldA-dependent adenosylation but are catalytically as competent as the wild-type protein when cob(I)alamin i provided as substrate, overview
Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
insoluble recombinant enzyme is lysed under anoxic conditions using CHES/glycine buffer, pH 9.5, followed by resuspension of EutT from the pelleted material with 8 mM CHAPS, and centrifugation
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solubilization of wild-type enzyme and truncated enzyme forms from inclusion bodies after expression in Escherichia coli
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APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
medicine
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insight in molecular mechanism of methylmalonic aciduria
medicine
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ACA-mediated catalysis provides insights in molecular basis for dysfunction in methylmalonic aciduria