Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Streptomyces clavuligerus |
Protein Variants | Comment | Organism |
---|---|---|
R258A | wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme | Streptomyces clavuligerus |
R258A | site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme | Streptomyces clavuligerus |
R258F | wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme | Streptomyces clavuligerus |
R258F | site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme | Streptomyces clavuligerus |
R258H | wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme | Streptomyces clavuligerus |
R258H | site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme | Streptomyces clavuligerus |
R258K | site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme | Streptomyces clavuligerus |
R258L | wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutant enzyme has broadened cosubstrate selectivity and is able to utilize hydrophobic 2-oxoacids. The efficiency of 2-oxoglutarate utilization is decreased as compared to the wild-type enzyme | Streptomyces clavuligerus |
R258L | site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme | Streptomyces clavuligerus |
R258Q | site-directed mutagenesis, broadened specificity for the cosubstrate compared to the wild-type enzyme, mutant enzyme can utilize hydrophobic 2-oxoacids, activity is decreased compared to the wild-type enzyme | Streptomyces clavuligerus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | KM-values for mutant enzymes with penicillin G or Ampicillin as prime substrate and 2-oxo-4-methylpentanoate, 2-oxoglutarate or 2-oxohexanoate as cosubstrate | Streptomyces clavuligerus | |
0.79 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258F | Streptomyces clavuligerus | |
0.79 | - |
penicillin G | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258A and R258Q | Streptomyces clavuligerus | |
0.84 | - |
penicillin G | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258F | Streptomyces clavuligerus | |
1.1 | - |
penicillin G | wild-type enzyme | Streptomyces clavuligerus | |
1.1 | - |
penicillin G | pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme | Streptomyces clavuligerus | |
1.2 | - |
penicillin G | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258L | Streptomyces clavuligerus | |
1.3 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258L | Streptomyces clavuligerus | |
1.5 | - |
penicillin G | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258H and R258Q | Streptomyces clavuligerus | |
1.6 | - |
penicillin G | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258F | Streptomyces clavuligerus | |
1.7 | - |
penicillin G | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L | Streptomyces clavuligerus | |
1.8 | - |
penicillin G | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258A | Streptomyces clavuligerus | |
1.9 | - |
ampicillin | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258F | Streptomyces clavuligerus | |
2.6 | - |
ampicillin | wild-type enzyme | Streptomyces clavuligerus | |
2.6 | - |
ampicillin | pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme | Streptomyces clavuligerus | |
2.6 | - |
ampicillin | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L | Streptomyces clavuligerus | |
3 | - |
penicillin G | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258H | Streptomyces clavuligerus | |
3.3 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q | Streptomyces clavuligerus | |
4.1 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H | Streptomyces clavuligerus | |
4.2 | - |
ampicillin | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258H and R258A | Streptomyces clavuligerus | |
4.5 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258A | Streptomyces clavuligerus | |
4.7 | - |
penicillin G | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K | Streptomyces clavuligerus | |
6.4 | - |
penicillin G | pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K | Streptomyces clavuligerus | |
6.6 | - |
ampicillin | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q | Streptomyces clavuligerus | |
7 | - |
penicillin G | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K | Streptomyces clavuligerus | |
13 | - |
ampicillin | pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K | Streptomyces clavuligerus | |
15 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K | Streptomyces clavuligerus | |
24 | - |
ampicillin | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K | Streptomyces clavuligerus |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
34500 | - |
x * 34500, about, recombinant wild-type and mutant enzymes, SDS-PAGE | Streptomyces clavuligerus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
penicillin N + 2-oxoglutarate + O2 | Streptomyces clavuligerus | key step in the cephamycin C biosynthesis pathway, the ring expansion step by incorporation of a methyl group into the cephem ring | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
ir |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Streptomyces clavuligerus | - |
recombinant enzyme expressed in Escherichia coli | - |
Streptomyces clavuligerus | P18548 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange chromatography and gel filtration | Streptomyces clavuligerus |
recombinant wild-type enzyme and mutant enzymes R258K, R258H, R258A, R258L and R258F | Streptomyces clavuligerus |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
activity with different substrates and assay methods of recombinant wild-type and mutant enzymes, overview | Streptomyces clavuligerus |
190 | - |
purified recombinant wild-type | Streptomyces clavuligerus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ampicillin + 2-oxo-4-methyl-pentanoic acid + O2 | mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme | Streptomyces clavuligerus | ? | - |
ir | |
ampicillin + 2-oxo-4-methylpentanoate + O2 | wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids | Streptomyces clavuligerus | cephalexin + succinate + CO2 + H2O | - |
? | |
ampicillin + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus | cephalexin + succinate + CO2 + H2O | - |
? | |
ampicillin + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus | ? | - |
ir | |
ampicillin + 2-oxohexanoate + O2 | wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids | Streptomyces clavuligerus | cephalexin + ? | - |
? | |
ampicillin + 2-oxohexanoic acid + O2 | mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme | Streptomyces clavuligerus | ? | - |
ir | |
penicillin G + 2-oxo-4-methyl-pentanoic acid + O2 | mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme | Streptomyces clavuligerus | phenylacetyl-7-aminodeacetoxycephalosporanic acid + 3-methylbutanoate + CO2 + H2O | - |
ir | |
penicillin G + 2-oxo-4-methylpentanoate + O2 | wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids | Streptomyces clavuligerus | phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ? | - |
? | |
penicillin G + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus | phenylacetyl-7-aminodeacetoxy cephalosporanic acid + succinate + CO2 + H2O | - |
? | |
penicillin G + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus | phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O | - |
ir | |
penicillin G + 2-oxohexanoate + O2 | wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids | Streptomyces clavuligerus | phenylacetyl-7-aminodeacetoxy cephalosporanic acid + ? | - |
? | |
penicillin G + 2-oxohexanoic acid + O2 | mutants R258H, R258K, R258Q, R258A, R258L, R258F, no activity with the wild-type enzyme | Streptomyces clavuligerus | phenylacetyl-7-aminodeacetoxycephalosporanic acid + pentanoate + CO2 + H2O | - |
ir | |
penicillin N + 2-oxoglutarate + O2 | wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids, the mutants R258A, R258L, R258H and R258F have broadened cosubstrate selectivity and are able to utilize hydrophobic 2-oxoacids | Streptomyces clavuligerus | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
? | |
penicillin N + 2-oxoglutarate + O2 | key step in the cephamycin C biosynthesis pathway, the ring expansion step by incorporation of a methyl group into the cephem ring | Streptomyces clavuligerus | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
ir | |
penicillin N + 2-oxoglutarate + O2 | specific for 2-oxoglutarate, which cannot be substituted by other 2-oxoacids e.g. 2-oxohexanoic acid, or 2-oxo-4-methyl-pentanoic acid | Streptomyces clavuligerus | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
ir |
Subunits | Comment | Organism |
---|---|---|
? | x * 34500, about, recombinant wild-type and mutant enzymes, SDS-PAGE | Streptomyces clavuligerus |
Synonyms | Comment | Organism |
---|---|---|
DAOCS | - |
Streptomyces clavuligerus |
deacetoxycephalosporin C synthase | - |
Streptomyces clavuligerus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.001 | - |
penicillin G | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K | Streptomyces clavuligerus | |
0.005 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H | Streptomyces clavuligerus | |
0.007 | - |
ampicillin | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258H | Streptomyces clavuligerus | |
0.008 | - |
ampicillin | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258A | Streptomyces clavuligerus | |
0.009 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258L | Streptomyces clavuligerus | |
0.009 | - |
ampicillin | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258K | Streptomyces clavuligerus | |
0.01 | - |
penicillin G | pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K | Streptomyces clavuligerus | |
0.01 | - |
penicillin G | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q | Streptomyces clavuligerus | |
0.011 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258A and R258F | Streptomyces clavuligerus | |
0.011 | - |
ampicillin | pH 7.5, cosubstrate 2-oxoglutarate, recombinant mutant R258K | Streptomyces clavuligerus | |
0.012 | - |
ampicillin | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258L and R258F | Streptomyces clavuligerus | |
0.013 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258K | Streptomyces clavuligerus | |
0.013 | - |
penicillin G | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutants R258A and R258H | Streptomyces clavuligerus | |
0.014 | - |
ampicillin | wild-type enzyme | Streptomyces clavuligerus | |
0.014 | - |
penicillin G | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258H | Streptomyces clavuligerus | |
0.014 | - |
ampicillin | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q | Streptomyces clavuligerus | |
0.014 | - |
ampicillin | pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme | Streptomyces clavuligerus | |
0.017 | - |
ampicillin | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258Q | Streptomyces clavuligerus | |
0.019 | - |
penicillin G | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258L | Streptomyces clavuligerus | |
0.02 | - |
penicillin G | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutants R258K, R258A, and R258L | Streptomyces clavuligerus | |
0.021 | - |
penicillin G | wild-type enzyme | Streptomyces clavuligerus | |
0.021 | - |
penicillin G | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258Q | Streptomyces clavuligerus | |
0.021 | - |
penicillin G | pH 7.5, cosubstrate 2-oxoglutarate, recombinant wild-type enzyme | Streptomyces clavuligerus | |
0.024 | - |
penicillin G | pH 7.5, cosubstrate 2-oxohexanoate, recombinant mutant R258F | Streptomyces clavuligerus | |
0.025 | - |
penicillin G | pH 7.5, cosubstrate 2-oxo-4-methyl-pentanoate, recombinant mutant R258F | Streptomyces clavuligerus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Streptomyces clavuligerus |