Cloned (Comment) | Organism |
---|---|
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 | Salmonella enterica |
Crystallization (Comment) | Organism |
---|---|
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 | Salmonella enterica |
Protein Variants | Comment | Organism |
---|---|---|
F91A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Salmonella enterica |
F91A,W93A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Salmonella enterica |
F91D | site-directed mutagenesis, the mutant is inactive | Salmonella enterica |
F91H | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Salmonella enterica |
F91W | site-directed mutagenesis, the mutant shows the same activity as the wild-type enzyme | Salmonella enterica |
F91W,W93F | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Salmonella enterica |
F91Y | site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme | Salmonella enterica |
W93A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Salmonella enterica |
W93D | site-directed mutagenesis, the mutant shows the same activity as the wild-type enzyme | Salmonella enterica |
W93F | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Salmonella enterica |
W93H | site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme | Salmonella enterica |
W93Y | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme | Salmonella enterica |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | kinetics for wild-type and mutants enzymes from two different assay methods, overview | Salmonella enterica | |
0.0003 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91Y | Salmonella enterica | |
0.001 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91Y | Salmonella enterica | |
0.002 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91A/W93D | Salmonella enterica | |
0.002 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93D | Salmonella enterica | |
0.002 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93H | Salmonella enterica | |
0.002 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93Y | Salmonella enterica | |
0.003 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91A/W93D | Salmonella enterica | |
0.003 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91H | Salmonella enterica | |
0.003 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91H | Salmonella enterica | |
0.003 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93A | Salmonella enterica | |
0.003 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93D | Salmonella enterica | |
0.004 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant wild-type CobA | Salmonella enterica | |
0.005 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91A | Salmonella enterica | |
0.005 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91W | Salmonella enterica | |
0.006 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91W/W93F | Salmonella enterica | |
0.007 | - |
ATP | pH 8.0, 25°C, recombinant wild-type CobA | Salmonella enterica | |
0.009 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91A | Salmonella enterica | |
0.009 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91W | Salmonella enterica | |
0.009 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91W/W93F | Salmonella enterica | |
0.011 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93A | Salmonella enterica | |
0.012 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93F | Salmonella enterica | |
0.016 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93Y | Salmonella enterica | |
0.026 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93H | Salmonella enterica | |
0.03 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93F | Salmonella enterica |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
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 | Salmonella enterica |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + cob(I)alamin | Salmonella enterica | - |
triphosphate + adenosylcob(I)alamine | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Salmonella enterica | - |
gene cobA | - |
Purification (Comment) | Organism |
---|---|
recombinant tagless, wild-type CobA, and His7-tagged mutant enzymes by nickel affinity chromatography, the His-tag is cleaved off by rTEV protease | Salmonella enterica |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
2 ATP + 2 cob(II)alamin + a reduced flavoprotein = 2 triphosphate + 2 adenosylcob(III)alamin + an oxidized flavoprotein | 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 | Salmonella enterica | |
2 ATP + 2 cob(II)yrinic acid a,c-diamide + a reduced flavoprotein = 2 triphosphate + 2 adenosylcob(III)yrinic acid a,c-diamide + an oxidized flavoprotein | 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 | Salmonella enterica |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + cob(I)alamin | - |
Salmonella enterica | triphosphate + adenosylcob(I)alamine | - |
? |
Synonyms | Comment | Organism |
---|---|---|
ACAT | - |
Salmonella enterica |
ATP:co(I)rrinoid adenosyltransferase | - |
Salmonella enterica |
CobA | - |
Salmonella enterica |
PduO | - |
Salmonella enterica |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Salmonella enterica |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.004 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91W/W93F | Salmonella enterica | |
0.008 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91A/W93D | Salmonella enterica | |
0.009 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93F | Salmonella enterica | |
0.009 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93Y | Salmonella enterica | |
0.01 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91A/W93D | Salmonella enterica | |
0.01 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91W/W93F | Salmonella enterica | |
0.01 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93F | Salmonella enterica | |
0.02 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91A | Salmonella enterica | |
0.02 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91A | Salmonella enterica | |
0.02 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91H | Salmonella enterica | |
0.02 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91H | Salmonella enterica | |
0.04 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93A | Salmonella enterica | |
0.05 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91Y | Salmonella enterica | |
0.05 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91Y | Salmonella enterica | |
0.05 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93A | Salmonella enterica | |
0.05 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93D | Salmonella enterica | |
0.05 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93D | Salmonella enterica | |
0.07 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93H | Salmonella enterica | |
0.08 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91W | Salmonella enterica | |
0.08 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91W | Salmonella enterica | |
0.08 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93H | Salmonella enterica | |
0.08 | - |
ATP | pH 8.0, 25°C, recombinant wild-type CobA | Salmonella enterica | |
0.08 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant wild-type CobA | Salmonella enterica | |
0.1 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93Y | Salmonella enterica |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Salmonella enterica |
General Information | Comment | Organism |
---|---|---|
evolution | ATP:co(I)rrinoid adenosyltransferases, ACATs, are separated into three families namely, CobA, EutT, and PduO | Salmonella enterica |
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 | Salmonella enterica |
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 | Salmonella enterica |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.3 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93F | Salmonella enterica | |
0.4 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91W/W93F | Salmonella enterica | |
0.6 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93Y | Salmonella enterica | |
0.8 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93F | Salmonella enterica | |
0.9 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91W | Salmonella enterica | |
2 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91A | Salmonella enterica | |
2 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91W/W93F | Salmonella enterica | |
3 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93H | Salmonella enterica | |
4 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91A | Salmonella enterica | |
4 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93A | Salmonella enterica | |
5 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91A/W93D | Salmonella enterica | |
5 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91A/W93D | Salmonella enterica | |
5 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91H | Salmonella enterica | |
6 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91H | Salmonella enterica | |
7 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93Y | Salmonella enterica | |
10 | - |
ATP | pH 8.0, 25°C, recombinant wild-type CobA | Salmonella enterica | |
20 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91W | Salmonella enterica | |
20 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93A | Salmonella enterica | |
20 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant W93D | Salmonella enterica | |
20 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93D | Salmonella enterica | |
20 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant wild-type CobA | Salmonella enterica | |
40 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant W93H | Salmonella enterica | |
50 | - |
cob(I)alamin | pH 8.0, 25°C, recombinant CobA mutant F91Y | Salmonella enterica | |
200 | - |
ATP | pH 8.0, 25°C, recombinant CobA mutant F91Y | Salmonella enterica |