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Literature summary for 2.5.1.17 extracted from

  • Moore, T.C.; Newmister, S.A.; Rayment, I.; Escalante-Semerena, J.C.
    Structural insights into the mechanism of four-coordinate Cob(II)alamin formation in the active site of the Salmonella enterica ATP:Co(I)rrinoid adenosyltransferase enzyme: critical role of residues Phe91 and Trp93 (2012), Biochemistry, 51, 9647-9657.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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 (Commentary)

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

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 [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

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/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + cob(I)alamin Salmonella enterica
-
triphosphate + adenosylcob(I)alamine
-
?

Organism

Organism UniProt Comment Textmining
Salmonella enterica
-
gene cobA
-

Purification (Commentary)

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

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 and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + cob(I)alamin
-
Salmonella enterica triphosphate + adenosylcob(I)alamine
-
?

Synonyms

Synonyms Comment Organism
ACAT
-
Salmonella enterica
ATP:co(I)rrinoid adenosyltransferase
-
Salmonella enterica
CobA
-
Salmonella enterica
PduO
-
Salmonella enterica

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Salmonella enterica

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Salmonella enterica

General Information

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 [mM/s]

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