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Literature summary 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)

EC Number Cloned (Comment) Organism
2.5.1.17 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)

EC Number Crystallization (Comment) Organism
2.5.1.17 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

EC Number Protein Variants Comment Organism
2.5.1.17 F91A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Salmonella enterica
2.5.1.17 F91A,W93A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Salmonella enterica
2.5.1.17 F91D site-directed mutagenesis, the mutant is inactive Salmonella enterica
2.5.1.17 F91H site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Salmonella enterica
2.5.1.17 F91W site-directed mutagenesis, the mutant shows the same activity as the wild-type enzyme Salmonella enterica
2.5.1.17 F91W,W93F site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Salmonella enterica
2.5.1.17 F91Y site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Salmonella enterica
2.5.1.17 W93A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Salmonella enterica
2.5.1.17 W93D site-directed mutagenesis, the mutant shows the same activity as the wild-type enzyme Salmonella enterica
2.5.1.17 W93F site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Salmonella enterica
2.5.1.17 W93H site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme Salmonella enterica
2.5.1.17 W93Y site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Salmonella enterica

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.5.1.17 additional information
-
 additional information kinetics for wild-type and mutants enzymes from two different assay methods, overview Salmonella enterica
2.5.1.17 0.0003
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91Y Salmonella enterica
2.5.1.17 0.001
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91Y Salmonella enterica
2.5.1.17 0.002
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91A/W93D Salmonella enterica
2.5.1.17 0.002
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93D Salmonella enterica
2.5.1.17 0.002
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93H Salmonella enterica
2.5.1.17 0.002
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93Y Salmonella enterica
2.5.1.17 0.003
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91A/W93D Salmonella enterica
2.5.1.17 0.003
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91H Salmonella enterica
2.5.1.17 0.003
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91H Salmonella enterica
2.5.1.17 0.003
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93A Salmonella enterica
2.5.1.17 0.003
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93D Salmonella enterica
2.5.1.17 0.004
-
 cob(I)alamin pH 8.0, 25°C, recombinant wild-type CobA Salmonella enterica
2.5.1.17 0.005
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91A Salmonella enterica
2.5.1.17 0.005
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91W Salmonella enterica
2.5.1.17 0.006
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91W/W93F Salmonella enterica
2.5.1.17 0.007
-
 ATP pH 8.0, 25°C, recombinant wild-type CobA Salmonella enterica
2.5.1.17 0.009
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91A Salmonella enterica
2.5.1.17 0.009
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91W Salmonella enterica
2.5.1.17 0.009
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91W/W93F Salmonella enterica
2.5.1.17 0.011
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93A Salmonella enterica
2.5.1.17 0.012
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93F Salmonella enterica
2.5.1.17 0.016
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93Y Salmonella enterica
2.5.1.17 0.026
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93H Salmonella enterica
2.5.1.17 0.03
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93F Salmonella enterica

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.5.1.17 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)

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

Organism

EC Number Organism UniProt Comment Textmining
2.5.1.17 Salmonella enterica
-
 gene cobA
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.5.1.17 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

EC Number Reaction Comment Organism Reaction ID
2.5.1.17 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
a
2.5.1.17 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
a

Substrates and Products (Substrate)

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

Synonyms

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.5.1.17 25
-
 assay at Salmonella enterica

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.5.1.17 0.004
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91W/W93F Salmonella enterica
2.5.1.17 0.008
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91A/W93D Salmonella enterica
2.5.1.17 0.009
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93F Salmonella enterica
2.5.1.17 0.009
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93Y Salmonella enterica
2.5.1.17 0.01
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91A/W93D Salmonella enterica
2.5.1.17 0.01
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91W/W93F Salmonella enterica
2.5.1.17 0.01
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93F Salmonella enterica
2.5.1.17 0.02
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91A Salmonella enterica
2.5.1.17 0.02
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91A Salmonella enterica
2.5.1.17 0.02
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91H Salmonella enterica
2.5.1.17 0.02
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91H Salmonella enterica
2.5.1.17 0.04
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93A Salmonella enterica
2.5.1.17 0.05
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91Y Salmonella enterica
2.5.1.17 0.05
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91Y Salmonella enterica
2.5.1.17 0.05
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93A Salmonella enterica
2.5.1.17 0.05
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93D Salmonella enterica
2.5.1.17 0.05
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93D Salmonella enterica
2.5.1.17 0.07
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93H Salmonella enterica
2.5.1.17 0.08
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91W Salmonella enterica
2.5.1.17 0.08
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91W Salmonella enterica
2.5.1.17 0.08
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93H Salmonella enterica
2.5.1.17 0.08
-
 ATP pH 8.0, 25°C, recombinant wild-type CobA Salmonella enterica
2.5.1.17 0.08
-
 cob(I)alamin pH 8.0, 25°C, recombinant wild-type CobA Salmonella enterica
2.5.1.17 0.1
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93Y Salmonella enterica

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.5.1.17 8
-
 assay at Salmonella enterica

General Information

EC Number General Information Comment Organism
2.5.1.17 evolution ATP:co(I)rrinoid adenosyltransferases, ACATs, are separated into three families namely, CobA, EutT, and PduO Salmonella enterica
2.5.1.17 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
2.5.1.17 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.5.1.17 0.3
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93F Salmonella enterica
2.5.1.17 0.4
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91W/W93F Salmonella enterica
2.5.1.17 0.6
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93Y Salmonella enterica
2.5.1.17 0.8
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93F Salmonella enterica
2.5.1.17 0.9
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91W Salmonella enterica
2.5.1.17 2
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91A Salmonella enterica
2.5.1.17 2
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91W/W93F Salmonella enterica
2.5.1.17 3
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93H Salmonella enterica
2.5.1.17 4
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91A Salmonella enterica
2.5.1.17 4
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93A Salmonella enterica
2.5.1.17 5
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91A/W93D Salmonella enterica
2.5.1.17 5
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91A/W93D Salmonella enterica
2.5.1.17 5
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91H Salmonella enterica
2.5.1.17 6
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91H Salmonella enterica
2.5.1.17 7
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93Y Salmonella enterica
2.5.1.17 10
-
 ATP pH 8.0, 25°C, recombinant wild-type CobA Salmonella enterica
2.5.1.17 20
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91W Salmonella enterica
2.5.1.17 20
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93A Salmonella enterica
2.5.1.17 20
-
 ATP pH 8.0, 25°C, recombinant CobA mutant W93D Salmonella enterica
2.5.1.17 20
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93D Salmonella enterica
2.5.1.17 20
-
 cob(I)alamin pH 8.0, 25°C, recombinant wild-type CobA Salmonella enterica
2.5.1.17 40
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant W93H Salmonella enterica
2.5.1.17 50
-
 cob(I)alamin pH 8.0, 25°C, recombinant CobA mutant F91Y Salmonella enterica
2.5.1.17 200
-
 ATP pH 8.0, 25°C, recombinant CobA mutant F91Y Salmonella enterica