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Literature summary for 6.2.1.14 extracted from
- The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes (2017), Nat. Chem. Biol., 13, 668-674 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene bioW, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta2 (DE3) | Aquifex aeolicus |
| gene bioW, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta2 (DE3) | Bacillus amyloliquefaciens |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| 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 |
| crystal structure determination and analysis of wild-type enzyme | Bacillus amyloliquefaciens |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| 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 |
| 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 |
| R201A | site-directed mutagenesis, the mutation has little effect on product formation | Aquifex aeolicus |
| R215A | site-directed mutagenesis, the mutant demonstrates a substantial reduction in product formation | Aquifex aeolicus |
| S182A | site-directed mutagenesis, the mutant demonstrates a substantial reduction in product formation | Aquifex aeolicus |
| 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 |
| Y199A | site-directed mutagenesis, the mutation has little effect on product formation | Aquifex aeolicus |
General Stability
| General Stability | Organism |
|---|---|
| propensity of the purified recombinant enzyme to precipitate and by the loss of activity upon prolonged incubation | Aquifex aeolicus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | kinetics | Aquifex aeolicus | |
| 0.0107 | - |
6-Carboxyhexanoate | pH 7.0, 37°C | Aquifex aeolicus |  |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Aquifex aeolicus | |
| Mg2+ | required | Bacillus amyloliquefaciens | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + 6-carboxyhexanoate + CoA | Aquifex aeolicus | - |
AMP + diphosphate + 6-carboxyhexanoyl-CoA | - |
? | |
| ATP + 6-carboxyhexanoate + CoA | Bacillus amyloliquefaciens | - |
AMP + diphosphate + 6-carboxyhexanoyl-CoA | - |
? | |
| ATP + 6-carboxyhexanoate + CoA | Bacillus amyloliquefaciens ATCC 23350 / DSM 7 / BCRC 11601 / NBRC 15535 / NRRL B-14393 | - |
AMP + diphosphate + 6-carboxyhexanoyl-CoA | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Aquifex aeolicus | O67575 | - |
- |
| Bacillus amyloliquefaciens | E1UV19 | - |
- |
| Bacillus amyloliquefaciens ATCC 23350 / DSM 7 / BCRC 11601 / NBRC 15535 / NRRL B-14393 | E1UV19 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| 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 |
| 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)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + 6-carboxyhexanoate + CoA | - |
Aquifex aeolicus | AMP + diphosphate + 6-carboxyhexanoyl-CoA | - |
? | |
| ATP + 6-carboxyhexanoate + CoA | - |
Bacillus amyloliquefaciens | AMP + diphosphate + 6-carboxyhexanoyl-CoA | - |
? | |
| ATP + 6-carboxyhexanoate + CoA | - |
Bacillus amyloliquefaciens ATCC 23350 / DSM 7 / BCRC 11601 / NBRC 15535 / NRRL B-14393 | AMP + diphosphate + 6-carboxyhexanoyl-CoA | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| More | primary sequence analysis | Aquifex aeolicus |
| More | primary sequence analysis | Bacillus amyloliquefaciens |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| AaBioW | - |
Aquifex aeolicus |
| BaBioW | - |
Bacillus amyloliquefaciens |
| BioW | - |
Aquifex aeolicus |
| BioW | - |
Bacillus amyloliquefaciens |
| Pimeloyl-CoA synthetase | - |
Aquifex aeolicus |
| Pimeloyl-CoA synthetase | - |
Bacillus amyloliquefaciens |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 37 | - |
assay at | Aquifex aeolicus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 7.45 | - |
6-Carboxyhexanoate | pH 7.0, 37°C | Aquifex aeolicus | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7 | - |
assay at | Aquifex aeolicus |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | - |
Aquifex aeolicus | |
| ATP | - |
Bacillus amyloliquefaciens | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | Aquifex aeolicus BioW represents a distinct protein fold within the superfamily of adenylating enzymes | Aquifex aeolicus |
| malfunction | BioW activity to hydrolyze adenylates of noncognate substrates can be abolished by mutation of a single residue, R159A | Aquifex aeolicus |
| additional information | structure-function relationship | Bacillus amyloliquefaciens |
| 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 |
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