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IUBMB CommentsThis enzyme is the first glutamine amidotransferase that participates in the anaerobic (early cobalt insertion) biosynthetic pathway of adenosylcobalamin, and catalyses the ATP-dependent synthesis of cobyrinate a,c-diamide from cobyrinate using either L-glutamine or ammonia as the nitrogen source. It is proposed that the enzyme first catalyses 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. The Km for ammonia is substantially higher than that for L-glutamine. The equivalent reaction in the aerobic cobalamin biosynthesis pathway is catalysed by EC 6.3.5.9, hydrogenobyrinic acid a,c-diamide synthase (glutamine-hydrolysing).
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2 ATP + cobyrinate + 2 L-glutamine + 2 H2O = 2 ADP + 2 phosphate + cobyrinate a,c-diamide + 2 L-glutamate
ATP + cobyrinate + L-glutamine + H2O = ADP + phosphate + cobyrinate c-monamide + L-glutamate
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ATP + cobyrinate c-monamide + L-glutamine + H2O = ADP + phosphate + cobyrinate a,c-diamide + L-glutamate
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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
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2 ATP + cobyrinate + 2 L-glutamine + 2 H2O = 2 ADP + 2 phosphate + cobyrinate a,c-diamide + 2 L-glutamate
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2 ATP + (aq)2cobyrinate + 2 L-glutamine + 2 H2O
2 ADP + 2 phosphate + (aq)2cobyrinate a,c-diamide + 2 L-glutamate
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76% of the initial velocity with cobyrinate
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2 ATP + (CN,aq)cobyrinate + 2 L-glutamine + 2 H2O
2 ADP + 2 phosphate + (CN,aq)cobyrinate a,c-diamide + 2 L-glutamate
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2 ATP + cobyrinate + 2 ammonia + 2 H2O
2 ADP + 2 phosphate + cobyrinate a,c-diamide
2 ATP + cobyrinate + 2 L-glutamine + 2 H2O
2 ADP + 2 phosphate + cobyrinate a,c-diamide + 2 L-glutamate
2 ATP + cobyrinate g-monoamide + L-glutamine + 2 H2O
2 ADP + phosphate + cobyrinate c,g-diamide + L-glutamate
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145% of the initial velocity with cobyrinate
product is 83% cobyrinate c,g-diamide, 17% cobyrinate a,c,g-triamide
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2 ATP + hydrogenobyrinate + 2 L-glutamine + 2 H2O
2 ADP + 2 phosphate + hydrogenobyrinate a,c-diamide + 2 L-glutamate
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ATP + (CN,aq)cobyrinate c-monoamide + L-glutamine + H2O
ADP + phosphate + (CN,aq)cobyrinate a,c-diamide + L-glutamate
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ATP + cobyrinate a-monoamide + L-glutamine + H2O
ADP + phosphate + cobyrinate a,c-diamide + L-glutamate
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81% of the initial velocity with cobyrinate
product is 100% cobyrinate a,c-diamide
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ATP + cobyrinate c-monoamide + L-glutamine + H2O
ADP + phosphate + cobyrinate a,c-diamide + L-glutamate
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19% of the initial velocity with cobyrinate
product is 100% cobyrinate a,c-diamide
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ATP + hydrogenobyrinate c-monoamide + L-glutamine + H2O
ADP + phosphate + hydrogenobyrinate a,c-diamide + L-glutamate
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19% of the initial velocity with cobyrinate
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additional information
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2 ATP + cobyrinate + 2 ammonia + 2 H2O
2 ADP + 2 phosphate + cobyrinate a,c-diamide
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2 ATP + cobyrinate + 2 ammonia + 2 H2O
2 ADP + 2 phosphate + cobyrinate a,c-diamide
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2 ATP + cobyrinate + 2 L-glutamine + 2 H2O
2 ADP + 2 phosphate + cobyrinate a,c-diamide + 2 L-glutamate
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2 ATP + cobyrinate + 2 L-glutamine + 2 H2O
2 ADP + 2 phosphate + cobyrinate a,c-diamide + 2 L-glutamate
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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
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additional information
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enzyme catalyzes ATP-dependent formation of the amide group using the ammonia nitrogen produced by the glutaminase domains. The reaction proceeds through a phosphoacyl intermediate, which is then subjected to the nucleophilic attack of the amino nitrogen, which leads to the formation of the amide bond and liberation of phosphate. Enzyme belongs to a family of ATP-dependent enzymes that also includes dethiobiotin synthetase and cobyric acid synthase CobQ. This enzyme family is also related to the MinD family of ATPases involved in regulation of cell division in bacteria and archaea. CobB and CobQ also contain unusual Triad family class I glutamine amidotransferase domains with conserved Cys and His residues, but lacking the Glu residue of the catalytic triad
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additional information
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no further amidation of cobyrinate a,c-diamide and cobyrinate a,c, g-triamide
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additional information
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enzyme catalyzes ATP-dependent formation of the amide group using the ammonia nitrogen produced by the glutaminase domains. The reaction proceeds through a phosphoacyl intermediate, which is then subjected to the nucleophilic attack of the amino nitrogen, which leads to the formation of the amide bond and liberation of phosphate. Enzyme belongs to a family of ATP-dependent enzymes that also includes dethiobiotin synthetase and cobyric acid synthase CobQ. This enzyme family is also related to the MinD family of ATPases involved in regulation of cell division in bacteria and archaea. CobB and CobQ also contain unusual Triad family class I glutamine amidotransferase domains with conserved Cys and His residues, but lacking the Glu residue of the catalytic triad
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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
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physiological function
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co-expression of the cobA gene from Propionibacterium freudenreichii and the cbiA, -C, -D, -E, -T, -F, -G, -H, -J, -K, -L, and -P genes from Salmonella enterica serovar typhimurium in Escherichia coli result in the production of cobyrinic acid a,c-diamide. Strains that have neither cbiP nor cbiA synthesized 1-desmethylcobyrinic acid even in the presence of cbiD, suggesting that CbiA and CbiP are necessary for CbiD activity
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D45N
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below 0.02% of the amidation activity of wild-type, no change in the order of amidation
D48N
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0.2% of the amidation activity of wild-type, no change in the order of amidation
D97N
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32.4% of the amidation activity of wild-type, no change in the order of amidation
E90Q
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below 0.02% of the amidation activity of wild-type, no change in the order of amidation
L47A
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10.6% of the amidation activity of wild-type, specific decrease in the affinity of the enzyme for the c-monoamide intermediate
Y46A
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3.8% of the amidation activity of wild-type, specific decrease in the affinity of the enzyme for the c-monoamide intermediate
additional information
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when the Bacillus megaterium cob operon is cloned into a plasmid and transformed into an Escherichia coli strain containing the Salmonella typhimurium cbiP, it confers upon the host strain the ability to make the cobyric acid de no.o. However, cobyric acid synthesis is observed only when the strain is grown anaerobically
additional information
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co-expression of the cobA gene from Propionibacterium freudenreichii and the cbiA, -C, -D, -E, -T, -F, -G, -H, -J, -K, -L, and -P genes from Salmonella enterica serovar typhimurium in Escherichia coli result in the production of cobyrinic acid a,c-diamide. Strains that have neither cbiP nor cbiA synthesized 1-desmethylcobyrinic acid even in the presence of cbiD, suggesting that CbiA and CbiP are necessary for CbiD activity
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synthesis
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co-expression of the cobA gene from Propionibacterium freudenreichii and the cbiA, -C, -D, -E, -T, -F, -G, -H, -J, -K, -L, and -P genes from Salmonella enterica serovar typhimurium in Escherichia coli result in the production of cobyrinic acid a,c-diamide. Strains that have neither cbiP nor cbiA synthesized 1-desmethylcobyrinic acid even in the presence of cbiD, suggesting that CbiA and CbiP are necessary for CbiD activity
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Debussche, L.; Thibaut, D.; Cameron, B.; Crouzet, J.; Blanche, F.
Purification and characterization of cobyrinic acid a,c-diamide synthase from Pseudomonas denitrificans
J. Bacteriol.
172
6239-6244
1990
Pseudomonas denitrificans (nom. rej.)
brenda
Galperin, M.Y.; Grishin, N.V.
The synthetase domains of cobalamin biosynthesis amidotransferases cobB and cobQ belong to a new family of ATP-dependent amidoligases, related to dethiobiotin synthetase
Proteins
41
238-247
2000
Pseudomonas denitrificans (nom. rej.) (P21632), Salmonella enterica subsp. enterica serovar Typhimurium (P29946)
brenda
Fresquet, V.; Williams, L.; Raushel, F.M.
Mechanism of cobyrinic acid a,c-diamide synthetase from Salmonella typhimurium LT2
Biochemistry
43
10619-10627
2004
Salmonella enterica subsp. enterica serovar Typhimurium
brenda
Raux, E.; Lanois, A.; Rambach, A.; Warren, M.J.; Thermes, C.
Cobalamin (vitamin B12) biosynthesis: functional characterization of the Bacillus megaterium cbi genes required to convert uroporphyrinogen III into cobyrinic acid a,c-diamide
Biochem. J.
335 (Pt 1)
167-173
1998
Priestia megaterium
brenda
Roessner, C.A.; Williams, H.J.; Scott, A.I.
Genetically engineered production of 1-desmethylcobyrinic acid, 1-desmethylcobyrinic acid a,c-diamide, and cobyrinic acid a,c-diamide in Escherichia coli implies a role for CbiD in C-1 methylation in the anaerobic pathway to cobalamin
J. Biol. Chem.
280
16748-16753
2005
Salmonella enterica
brenda