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Literature summary for 6.2.1.14 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
Search for more literature for 6.2.1.14

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment) Organism
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

Protein Variants Comment Organism
R213A site-directed mutagenesis, almost inactive mutant Bacillus subtilis
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
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
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
Y211F site-directed mutagenesis, almost inactive mutant Bacillus subtilis
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
Y211F site-directed mutagenesis, the mutant retains 36% activity compared to wild-type Bacillus subtilis

KM Value [mM]

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

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Bacillus subtilis

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Bacillus subtilis P53559
-
-
Bacillus subtilis 168 P53559
-
-

Purification (Commentary)

Purification (Comment) Organism
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

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

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + 6-carboxyhexanoate + CoA
-
 Bacillus subtilis AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
 ?
ATP + 6-carboxyhexanoate + CoA best substrate Bacillus subtilis AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
 ?
ATP + 6-carboxyhexanoate + CoA
-
 Bacillus subtilis 168 AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
 ?
ATP + adipate + CoA
-
 Bacillus subtilis AMP + diphosphate + adipyl-CoA
-
 ?
ATP + azelate + CoA
-
 Bacillus subtilis AMP + diphosphate + azelayl-CoA
-
 ?
ATP + glutarate + CoA
-
 Bacillus subtilis AMP + diphosphate + glutaryl-CoA
-
 ?
ATP + glutarate + CoA
-
 Bacillus subtilis 168 AMP + diphosphate + glutaryl-CoA
-
 ?
ATP + heptanoate + CoA
-
 Bacillus subtilis AMP + diphosphate + heptanoyl-CoA
-
 ?
ATP + heptanoate + CoA
-
 Bacillus subtilis 168 AMP + diphosphate + heptanoyl-CoA
-
 ?
ATP + octanoate + CoA
-
 Bacillus subtilis AMP + diphosphate + octanoyl-CoA
-
 ?
ATP + octanoate + CoA
-
 Bacillus subtilis 168 AMP + diphosphate + octanoyl-CoA
-
 ?
ATP + suberate + CoA
-
 Bacillus subtilis AMP + diphosphate + suberyl-CoA
-
 ?
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 ?
-
 ?
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

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

Synonyms

Synonyms Comment Organism
BioW
-
 Bacillus subtilis
Pimeloyl-CoA synthetase
-
 Bacillus subtilis

Turnover Number [1/s]

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

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Bacillus subtilis

General Information

General Information Comment Organism
additional information residues controlling substrate binding and catalysis include Tyr199, Tyr211, Arg213 and Arg227 Bacillus subtilis
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