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Literature summary extracted from

  • Wang, M.; Moynie, L.; Harrison, P.J.; Kelly, V.; Piper, A.; Naismith, J.H.; Campopiano, D.J.
    Using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters (2017), Nat. Chem. Biol., 13, 660-667 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.2.1.14 gene bioW, sequence comparisons, recombinant expression of His6-tagged enzyme in Escherichia coli Bacillus subtilis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
6.2.1.14 purified K2PtCl4-pimeloyl-adenylate-diphosphate complex, pimelic acid-CoASH complex, pimeloyl-adenylate-diphosphate, and AMP-PNP-pimelic acid complexes, X-ray diffraction structure determination and analysis at 2.04-2.44 A resolution Bacillus subtilis

Protein Variants

EC Number Protein Variants Comment Organism
6.2.1.14 R213A site-directed mutagenesis, almost inactive mutant Bacillus subtilis
6.2.1.14 R227E site-directed mutagenesis, the mutant shows a turnover with the natural substrate pimelic acid that is reduced by around 25fold to about 4% activity remaining compared to the wild-type Bacillus subtilis
6.2.1.14 R227K site-directed mutagenesis, the mutant shows a turnover with the natural substrate pimelic acid that is reduced by around 25fold to about 4% activity remaining compared to the wild-type Bacillus subtilis
6.2.1.14 Y199F site-directed mutagenesis, the mutant retains 55% activity compared to wild-type, the Y199F mutant is inactive with heptanoic acid and octanoic acid, the Y199F mutant displayed a twofold greater activity with pimelic acid but no improvement with azelaic acid compared to wild-type Bacillus subtilis
6.2.1.14 Y211F site-directed mutagenesis, almost inactive mutant Bacillus subtilis
6.2.1.14 Y211F site-directed mutagenesis, the mutant displays activity with both monocarboxylic acid substrates, heptanoic acid and octanoic acid, the Y211F mutant displays about 4fold increased activity with the suberic acid substrate and 3fold increased activity with the azelaic acid substrate relative to the wild-type BioW. The mutant enzymes is also active with 7-bromoheptanoic acid, 7-aminoheptanoic acid, 6-methylheptanoic acid, 7-phenyl­heptanoic acid, and 7-octenoic acid, but not with 7-aminoheptanoic acid Bacillus subtilis
6.2.1.14 Y211F site-directed mutagenesis, the mutant retains 36% activity compared to wild-type Bacillus subtilis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.2.1.14 additional information
-
 additional information Michaelis-Menten enzyme kinetics, recombinant enzyme Bacillus subtilis
6.2.1.14 0.0705
-
 6-Carboxyhexanoate pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.2293
-
 CoA pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.2996
-
 ATP pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.4794
-
 Octanoate mutant Y211F, pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.529
-
 Heptanoate mutant Y211F, pH and temperature not specified in the publication Bacillus subtilis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.2.1.14 Mg2+ required Bacillus subtilis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.2.1.14 ATP + 6-carboxyhexanoate + CoA Bacillus subtilis
-
 AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
 ?
6.2.1.14 ATP + 6-carboxyhexanoate + CoA Bacillus subtilis 168
-
 AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.2.1.14 Bacillus subtilis P53559
-
-
6.2.1.14 Bacillus subtilis 168 P53559
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.2.1.14 recombinant His6-tagged enzyme by nickel affinity chromatography, removal of the tag by TEV protease, another step of nickel affinity chromatography, and gel filtration, to homogeneity Bacillus subtilis

Reaction

EC Number Reaction Comment Organism Reaction ID
6.2.1.14 ATP + 6-carboxyhexanoate + CoA = AMP + diphosphate + 6-carboxyhexanoyl-CoA strutcure-function analysis Bacillus subtilis
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.2.1.14 ATP + 6-carboxyhexanoate + CoA
-
 Bacillus subtilis AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
 ?
6.2.1.14 ATP + 6-carboxyhexanoate + CoA best substrate Bacillus subtilis AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
 ?
6.2.1.14 ATP + 6-carboxyhexanoate + CoA
-
 Bacillus subtilis 168 AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
 ?
6.2.1.14 ATP + adipate + CoA
-
 Bacillus subtilis AMP + diphosphate + adipyl-CoA
-
 ?
6.2.1.14 ATP + azelate + CoA
-
 Bacillus subtilis AMP + diphosphate + azelayl-CoA
-
 ?
6.2.1.14 ATP + glutarate + CoA
-
 Bacillus subtilis AMP + diphosphate + glutaryl-CoA
-
 ?
6.2.1.14 ATP + glutarate + CoA
-
 Bacillus subtilis 168 AMP + diphosphate + glutaryl-CoA
-
 ?
6.2.1.14 ATP + heptanoate + CoA
-
 Bacillus subtilis AMP + diphosphate + heptanoyl-CoA
-
 ?
6.2.1.14 ATP + heptanoate + CoA
-
 Bacillus subtilis 168 AMP + diphosphate + heptanoyl-CoA
-
 ?
6.2.1.14 ATP + octanoate + CoA
-
 Bacillus subtilis AMP + diphosphate + octanoyl-CoA
-
 ?
6.2.1.14 ATP + octanoate + CoA
-
 Bacillus subtilis 168 AMP + diphosphate + octanoyl-CoA
-
 ?
6.2.1.14 ATP + suberate + CoA
-
 Bacillus subtilis AMP + diphosphate + suberyl-CoA
-
 ?
6.2.1.14 additional information using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters. Azelaic acid is a poor substrate for BioW, substrate specificity and binding structures, overview Bacillus subtilis ?
-
 ?
6.2.1.14 additional information using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters. Azelaic acid is a poor substrate for BioW, substrate specificity and binding structures, overview Bacillus subtilis 168 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
6.2.1.14 homodimer secondary and tertiary structure determination and analysis of BioW, overview Bacillus subtilis

Synonyms

EC Number Synonyms Comment Organism
6.2.1.14 BioW
-
 Bacillus subtilis
6.2.1.14 Pimeloyl-CoA synthetase
-
 Bacillus subtilis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.2.1.14 0.0001
-
 azelate pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.0038
-
 suberate pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.0051
-
 Glutarate pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.0066
-
 adipate pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.008
-
 Octanoate mutant Y211F, pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.012
-
 Heptanoate mutant Y211F, pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.44
-
 ATP pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.48
-
 6-Carboxyhexanoate pH and temperature not specified in the publication Bacillus subtilis
6.2.1.14 0.87
-
 CoA pH and temperature not specified in the publication Bacillus subtilis

Cofactor

EC Number Cofactor Comment Organism Structure
6.2.1.14 ATP
-
 Bacillus subtilis

General Information

EC Number General Information Comment Organism
6.2.1.14 additional information residues controlling substrate binding and catalysis include Tyr199, Tyr211, Arg213 and Arg227 Bacillus subtilis
6.2.1.14 physiological function biotin is an essential vitamin in plants and mammals, functioning as the carbon dioxide carrier within central lipid metabo­lism. Bacterial pimeloyl-CoA synthetase (BioW) acts as a highly specific substrate-selection gate, ensuring the integrity of the carbon chain in biotin synthesis. BioW catalyzes the condensation of pimelic acid (C7 dicarboxylic acid) with CoASH in an ATP-dependent manner to form pimeloyl-CoA, the first dedicated biotin building block Bacillus subtilis