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Literature summary extracted from

  • Fresquet, V.; Williams, L.; Raushel, F.M.
    Mechanism of cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium LT2 (2004), Biochemistry, 43, 10619-10627.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.3.5.9 expression of wild-type and mutant enzymes in Escherichia coli Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 expression in Escherichia coli Salmonella enterica subsp. enterica serovar Typhimurium

Protein Variants

EC Number Protein Variants Comment Organism
6.3.5.9 D45N less than 0.2% of amidation activity with glutamine and with NH4+, compared to wild-type values Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 D48N less than 0.3% of amidation activity with glutamine and with NH4+, compared to wild-type values Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 D97N 32.4% of wild-type amidation activity with glutamine, 16.5% of wild-type amidation activity with NH4+ Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 E90Q less than 0.02% of amidation activity with glutamine and with NH4+, compared to wild-type values Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 L47A 10.6% of wild-type amidation activity with glutamine, 44% of wild-type amidation activity with NH4+, mutation specifically decreases the affinity of the enzyme to the cobyrinic acid c-monoamide Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 Y46A 3.8% of wild-type amidation activity with glutamine, 2% of wild-type amidation activity with NH4+, mutation specifically decreases the affinity of the enzyme to the cobyrinic acid c-monoamide Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 D45N below 0.02% of the amidation activity of wild-type, no change in the order of amidation Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 D48N 0.2% of the amidation activity of wild-type, no change in the order of amidation Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 D97N 32.4% of the amidation activity of wild-type, no change in the order of amidation Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 E90Q below 0.02% of the amidation activity of wild-type, no change in the order of amidation Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 L47A 10.6% of the amidation activity of wild-type, specific decrease in the affinity of the enzyme for the c-monoamide intermediate Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 Y46A 3.8% of the amidation activity of wild-type, specific decrease in the affinity of the enzyme for the c-monoamide intermediate Salmonella enterica subsp. enterica serovar Typhimurium

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.3.5.9 0.0003
-
 cobyrinic acid c-monoamide mutant enzyme D97N Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.00041
-
 cobyrinic acid c-monoamide wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.00074
-
 cobyrinic acid pH 7.7, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.0027
-
 cobyrinic acid c-monoamide mutant enzyme L47A Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.0027
-
 ATP pH 7.7, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.011
-
 cobyrinic acid c-monoamide mutant enzyme Y46A Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.053
-
 glutamine pH 7.7, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 26.2
-
 NH4+ pH 7.7, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 0.00074
-
 cobyrinate pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 0.0027
-
 ATP pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 0.053
-
 L-glutamine pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 26.2
-
 ammonia pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
6.3.5.9 50000
-
 1 * 50000, SDS-PAGE Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 55000
-
 gel filtration Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 50000
-
 1 * 50039, calculated, x * 50000, SDS-PAGE Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 50039
-
 1 * 50039, calculated, x * 50000, SDS-PAGE Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 53000
-
 gel filtration Salmonella enterica subsp. enterica serovar Typhimurium

Organism

EC Number Organism UniProt Comment Textmining
6.3.5.9 Salmonella enterica subsp. enterica serovar Typhimurium
-
-
-
6.3.5.11 Salmonella enterica subsp. enterica serovar Typhimurium
-
 LT2
-

Reaction

EC Number Reaction Comment Organism Reaction ID
6.3.5.11 2 ATP + cobyrinate + 2 L-glutamine + 2 H2O = 2 ADP + 2 phosphate + cobyrinate a,c-diamide + 2 L-glutamate catalytic mechanism in which CbiA catalyzes the amidation of the c-carboxylate, and then the intermediate is released into solution and binds to the same catalytic site for the amidation of the a-carboxylate Salmonella enterica subsp. enterica serovar Typhimurium
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.3.5.9 cobyrinic acid + ATP + glutamine model for catalytic mechanism in which CbiA catalyzes the amidation of the c-carboxylate and then the intermediate is released into solution and binds to the same catalytic site for the amidation of the a-carboxylate Salmonella enterica subsp. enterica serovar Typhimurium cobyrinic acid a,c-diamide + ADP + phosphate + glutamate
-
 ?
6.3.5.9 cobyrinic acid + ATP + NH4+ model for catalytic mechanism in which CbiA catalyzes the amidation of the c-carboxylate and then the intermediate is released into solution and binds to the same catalytic site for the amidation of the a-carboxylate Salmonella enterica subsp. enterica serovar Typhimurium cobyrinic acid a,c-diamide + ADP + phosphate
-
 ?
6.3.5.9 cobyrinic acid c-monoamide + ATP + glutamine
-
 Salmonella enterica subsp. enterica serovar Typhimurium cobyrinic acid a,c-diamide + ADP + phosphate + glutamate
-
 ?
6.3.5.9 cobyrinic acid c-monoamide + ATP + NH4+
-
 Salmonella enterica subsp. enterica serovar Typhimurium cobyrinic acid a,c-diamide + ADP + phosphate
-
 ?
6.3.5.11 2 ATP + cobyrinate + 2 ammonia + 2 H2O
-
 Salmonella enterica subsp. enterica serovar Typhimurium 2 ADP + 2 phosphate + cobyrinate a,c-diamide
-
 ?
6.3.5.11 2 ATP + cobyrinate + 2 L-glutamine + 2 H2O
-
 Salmonella enterica subsp. enterica serovar Typhimurium 2 ADP + 2 phosphate + cobyrinate a,c-diamide + 2 L-glutamate the hydrolysis of glutamine and the synthesis of the cobyrinic acid a,c-diamide product are uncoupled. CbiA catalyzes the sequential amidation of the c- and a-carboxylate groups of cobyrinic acid via the formation of a phosphorylated intermediate. CbiA catalyzes the amidation of the c-carboxylate, and then the intermediate is released into solution and binds to the same catalytic site for the amidation of the a-carboxylate ?
6.3.5.11 additional information CbiA is able to hydrolyze glutamine in the absence of the other two substrates. The addition of ATP alone decreases the Km for glutamine by a factor of 2. Cobyrinic acid enhances the kcat for the hydrolysis of glutamine about 2fold and decreases the Km for glutamine by 20fold. The decrease in the Km for glutamine is about 200fold when both ATP and cobyrinic acid are present in the assay solution. There is no change in the Km for glutamine when ADP is added with cobyrinic acid Salmonella enterica subsp. enterica serovar Typhimurium ?
-
 ?

Subunits

EC Number Subunits Comment Organism
6.3.5.9 monomer 1 * 50000, SDS-PAGE Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 monomer 1 * 50039, calculated, x * 50000, SDS-PAGE Salmonella enterica subsp. enterica serovar Typhimurium

Synonyms

EC Number Synonyms Comment Organism
6.3.5.9 CbiA
-
 Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 CbiA
-
 Salmonella enterica subsp. enterica serovar Typhimurium

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.3.5.9 0.008
-
 cobyrinic acid c-monoamide mutant enzyme Y46A Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.04
-
 cobyrinic acid c-monoamide mutant enzyme D97N Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.057
-
 cobyrinic acid c-monoamide mutant enzyme L47A Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.13
-
 ATP pH 7.7, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.16
-
 glutamine pH 7.7, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.16
-
 cobyrinic acid pH 7.7, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.18
-
 NH4+ pH 7.7, wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.9 0.2
-
 cobyrinic acid c-monoamide wild-type enzyme Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 0.13
-
 ATP pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 0.16
-
 L-glutamine pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 0.16
-
 cobyrinate pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 0.18
-
 ammonia pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.3.5.9 6.8 8
-
 Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 6.8 8
-
 Salmonella enterica subsp. enterica serovar Typhimurium

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
6.3.5.11 3.04
-
 L-glutamine pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 6.9
-
 ammonia pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 48.2
-
 ATP pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium
6.3.5.11 216
-
 cobyrinate pH 7.7, 30°C Salmonella enterica subsp. enterica serovar Typhimurium