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Literature summary extracted from

  • Estrada, P.; Manandhar, M.; Dong, S.H.; Deveryshetty, J.; Agarwal, V.; Cronan, J.E.; Nair, S.K.
    The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes (2017), Nat. Chem. Biol., 13, 668-674 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.2.1.14 gene bioW, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta2 (DE3) Aquifex aeolicus
6.2.1.14 gene bioW, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta2 (DE3) Bacillus amyloliquefaciens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
6.2.1.14 crystal structure determination and analysis of free wild-type AaBioW, selenomethinonine-labeled AaBioW with pimelate, AaBioW with AMP-CPP-Mg2+-pimelate, and AaBioW with CoA-AMP Aquifex aeolicus
6.2.1.14 crystal structure determination and analysis of wild-type enzyme Bacillus amyloliquefaciens

Protein Variants

EC Number Protein Variants Comment Organism
6.2.1.14 H16A site-directed mutagenesis, the mutant variant displays only a modest 20% loss in activity relative to the wild-type, reflecting the importance of these other interacting residues in stabilizing CoA binding Aquifex aeolicus
6.2.1.14 R159A site-directed mutagenesis, the activity to hydrolyze adenylates of noncognate substrates is abolished in the mutant. The R159A variant can no longer proofread, but the enzyme still retains ligase activity and can catalyze the formation of pimeloyl-CoA, the mutant demonstrates a notable reduction in turnover, which is in line with the function of the residue in forming the exterior wall of the pimelate-binding cavity Aquifex aeolicus
6.2.1.14 R201A site-directed mutagenesis, the mutation has little effect on product formation Aquifex aeolicus
6.2.1.14 R215A site-directed mutagenesis, the mutant demonstrates a substantial reduction in product formation Aquifex aeolicus
6.2.1.14 S182A site-directed mutagenesis, the mutant demonstrates a substantial reduction in product formation Aquifex aeolicus
6.2.1.14 Y187A site-directed mutagenesis, the mutant demonstrates a notable reduction in turnover, which is in line with the function of the residue in forming the exterior wall of the pimelate-binding cavity Aquifex aeolicus
6.2.1.14 Y199A site-directed mutagenesis, the mutation has little effect on product formation Aquifex aeolicus

General Stability

EC Number General Stability Organism
6.2.1.14 propen­sity of the purified recombinant enzyme to precipitate and by the loss of activity upon prolonged incubation Aquifex aeolicus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.2.1.14 additional information
-
additional information kinetics Aquifex aeolicus
6.2.1.14 0.0107
-
6-Carboxyhexanoate pH 7.0, 37°C Aquifex aeolicus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.2.1.14 Mg2+ required Aquifex aeolicus
6.2.1.14 Mg2+ required Bacillus amyloliquefaciens

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 Aquifex aeolicus
-
AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
?
6.2.1.14 ATP + 6-carboxyhexanoate + CoA Bacillus amyloliquefaciens
-
AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
?
6.2.1.14 ATP + 6-carboxyhexanoate + CoA Bacillus amyloliquefaciens ATCC 23350 / DSM 7 / BCRC 11601 / NBRC 15535 / NRRL B-14393
-
AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
?

Organism

EC Number Organism UniProt Comment Textmining
6.2.1.14 Aquifex aeolicus O67575
-
-
6.2.1.14 Bacillus amyloliquefaciens E1UV19
-
-
6.2.1.14 Bacillus amyloliquefaciens ATCC 23350 / DSM 7 / BCRC 11601 / NBRC 15535 / NRRL B-14393 E1UV19
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.2.1.14 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta2 (DE3) by nickel affinity chromatography, tag cleavage through thrombin, and gel filtration Aquifex aeolicus
6.2.1.14 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta2 (DE3) by nickel affinity chromatography, tag cleavage through thrombin, and gel filtration Bacillus amyloliquefaciens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.2.1.14 ATP + 6-carboxyhexanoate + CoA
-
Aquifex aeolicus AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
?
6.2.1.14 ATP + 6-carboxyhexanoate + CoA
-
Bacillus amyloliquefaciens AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
?
6.2.1.14 ATP + 6-carboxyhexanoate + CoA
-
Bacillus amyloliquefaciens ATCC 23350 / DSM 7 / BCRC 11601 / NBRC 15535 / NRRL B-14393 AMP + diphosphate + 6-carboxyhexanoyl-CoA
-
?

Subunits

EC Number Subunits Comment Organism
6.2.1.14 More primary sequence analysis Aquifex aeolicus
6.2.1.14 More primary sequence analysis Bacillus amyloliquefaciens

Synonyms

EC Number Synonyms Comment Organism
6.2.1.14 AaBioW
-
Aquifex aeolicus
6.2.1.14 BaBioW
-
Bacillus amyloliquefaciens
6.2.1.14 BioW
-
Aquifex aeolicus
6.2.1.14 BioW
-
Bacillus amyloliquefaciens
6.2.1.14 Pimeloyl-CoA synthetase
-
Aquifex aeolicus
6.2.1.14 Pimeloyl-CoA synthetase
-
Bacillus amyloliquefaciens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.2.1.14 37
-
assay at Aquifex aeolicus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.2.1.14 7.45
-
6-Carboxyhexanoate pH 7.0, 37°C Aquifex aeolicus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.2.1.14 7
-
assay at Aquifex aeolicus

Cofactor

EC Number Cofactor Comment Organism Structure
6.2.1.14 ATP
-
Aquifex aeolicus
6.2.1.14 ATP
-
Bacillus amyloliquefaciens

General Information

EC Number General Information Comment Organism
6.2.1.14 evolution Aquifex aeolicus BioW represents a distinct protein fold within the superfamily of adenylating enzymes Aquifex aeolicus
6.2.1.14 malfunction BioW activity to hydrolyze adenylates of noncognate substrates can be abolished by mutation of a single residue, R159A Aquifex aeolicus
6.2.1.14 additional information structure-function relationship Bacillus amyloliquefaciens
6.2.1.14 additional information substrate-bound structures are determined to identify the enzyme active site and elucidate the mechanistic strategy for conjugating CoA to the seven-carbon alpha,omega-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is remi­niscent of pre-transfer proofreading observed in some tRNA synthetases. BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of another protein fold. The movement of Arg159, which serves to position this residue to assist in thioester formation, is reminiscent of the domain movement in acetyl-CoA synthetase that positions a catalytic lysine important for adenylation away from the active site to facilitate thioester formation Aquifex aeolicus