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Literature summary for 1.14.20.1 extracted from
- New strategy of site-directed mutagenesis identifies new sites to improve Streptomyces clavuligerus deacetoxycephalosporin C synthase activity toward penicillin G (2012), Appl. Microbiol. Biotechnol., 93, 2395-2401.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression of wild-type and mutant enzymes in Escherichia coli strain BL21 | Streptomyces clavuligerus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| A61E | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| C37S | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| additional information | since the natural substrate of the enzyme, penicillin N, is not available in large quantities, engineering of a scDAOCS capable of converting the cheap and easily available substrate penicillin G is valuable for large-scale production of 7-aminodeacetoxycephalosporanic acid, 7-ADCA. Ten sites, Y184, V245, S261, C37, T42, V51, S59, A61, Q126, and T213, are mutated to 21 point mutants | Streptomyces clavuligerus |
| Q126M | site-directed mutagenesis, the mutant shows activity increased to 281.5% with penicillin G compared to the wild-type | Streptomyces clavuligerus |
| S261A | site-directed mutagenesis, the mutant shows activity increased to 128.8% with penicillin G compared to the wild-type | Streptomyces clavuligerus |
| S261I | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| S261L | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| S261M | site-directed mutagenesis, the mutant shows activity increased to 158.4% with penicillin G compared to the wild-type | Streptomyces clavuligerus |
| S261V | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| S59T | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| T213V | site-directed mutagenesis, the mutant shows activity increased to 172.4% with penicillin G compared to the wild-type | Streptomyces clavuligerus |
| T42A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| V245H | site-directed mutagenesis, inactive mutant | Streptomyces clavuligerus |
| V245K | site-directed mutagenesis, inactive mutant | Streptomyces clavuligerus |
| V245R | site-directed mutagenesis, inactive mutant | Streptomyces clavuligerus |
| V51A | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| Y184A | site-directed mutagenesis, the mutant shows activity increased to 143.9% with penicillin G compared to the wild-type | Streptomyces clavuligerus |
| Y184F | site-directed mutagenesis, inactive mutant | Streptomyces clavuligerus |
| Y184I | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| Y184L | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| Y184M | site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type | Streptomyces clavuligerus |
| Y184V | site-directed mutagenesis, inactive mutant | Streptomyces clavuligerus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 4.71 | - |
penicillin G | recombinant mutant Y184A, pH not specified in the publication, 30°C | Streptomyces clavuligerus |  |
| 5.28 | - |
penicillin G | recombinant mutant S261A, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 9.9 | - |
penicillin G | recombinant mutant T213V, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 14.38 | - |
penicillin G | recombinant wild-type enzyme, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 41.16 | - |
penicillin G | recombinant mutant Q126M, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 48.1 | - |
penicillin G | recombinant mutant S261M, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Iron | dependent on | Streptomyces clavuligerus | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | Streptomyces clavuligerus | Streptomyces clavuligerus deacetoxycephalosporin C synthase is an iron- and 2-ketoglutarate-dependent oxidase, with the ability to catalyze the ring expansion reaction of penicillins, which converts the thiazolidine ring in penicillins to the dihydrothiazine ring. scDAOCS converts its natural substrate, penicillin N, whereas it has very low activity on the bulk and cheap penicillins such as penicillin G | ? | - |
? | |
| additional information | Streptomyces clavuligerus ATCC 27064 | Streptomyces clavuligerus deacetoxycephalosporin C synthase is an iron- and 2-ketoglutarate-dependent oxidase, with the ability to catalyze the ring expansion reaction of penicillins, which converts the thiazolidine ring in penicillins to the dihydrothiazine ring. scDAOCS converts its natural substrate, penicillin N, whereas it has very low activity on the bulk and cheap penicillins such as penicillin G | ? | - |
? | |
| penicillin N + 2-oxoglutarate + O2 | Streptomyces clavuligerus | the enzyme catalyzes the ring expansion of penicillin substrates into a ring-expanded product to eventually produce cephamycin C | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
? | |
| penicillin N + 2-oxoglutarate + O2 | Streptomyces clavuligerus ATCC 27064 | the enzyme catalyzes the ring expansion of penicillin substrates into a ring-expanded product to eventually produce cephamycin C | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Streptomyces clavuligerus | - |
- |
- |
| Streptomyces clavuligerus ATCC 27064 | - |
- |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| soluble recombinant wild-type and mutant enzymes from Escherichia coli strain BL21 by anion exchange chromatography and gel filtration | Streptomyces clavuligerus |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 11.89 | - |
recombinant mutant S261I, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 13.44 | - |
recombinant mutant C37S, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 16.87 | - |
recombinant mutant T42A, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 20.03 | - |
recombinant mutant A61E, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 22.06 | - |
recombinant mutant S261V, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 30.86 | - |
recombinant mutant S261L, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 31.38 | - |
recombinant mutant Y184L, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 33.51 | - |
recombinant mutant Y184M, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 36.12 | - |
recombinant mutant Y184I, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 49.12 | - |
recombinant mutant S59T, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 56.84 | - |
recombinant wild-type enzyme, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 71.7 | - |
recombinant mutant Q126M, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 80.68 | - |
recombinant mutant T213V, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 90.04 | - |
recombinant mutant S261M, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 98 | - |
recombinant mutant S261A, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
| 160 | - |
recombinant mutant Y184A, pH not specified in the publication, 30°C | Streptomyces clavuligerus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | Streptomyces clavuligerus deacetoxycephalosporin C synthase is an iron- and 2-ketoglutarate-dependent oxidase, with the ability to catalyze the ring expansion reaction of penicillins, which converts the thiazolidine ring in penicillins to the dihydrothiazine ring. scDAOCS converts its natural substrate, penicillin N, whereas it has very low activity on the bulk and cheap penicillins such as penicillin G | Streptomyces clavuligerus | ? | - |
? | |
| additional information | Streptomyces clavuligerus deacetoxycephalosporin C synthase is an iron- and 2-ketoglutarate-dependent oxidase, with the ability to catalyze the ring expansion reaction of penicillins, which converts the thiazolidine ring in penicillins to the dihydrothiazine ring. scDAOCS converts its natural substrate, penicillin N, whereas it has very low activity on the bulk and cheap penicillins such as penicillin G | Streptomyces clavuligerus ATCC 27064 | ? | - |
? | |
| penicillin G + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus | phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O | - |
? | |
| penicillin G + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus ATCC 27064 | phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O | - |
? | |
| penicillin N + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
? | |
| penicillin N + 2-oxoglutarate + O2 | the enzyme catalyzes the ring expansion of penicillin substrates into a ring-expanded product to eventually produce cephamycin C | Streptomyces clavuligerus | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
? | |
| penicillin N + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus ATCC 27064 | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
? | |
| penicillin N + 2-oxoglutarate + O2 | the enzyme catalyzes the ring expansion of penicillin substrates into a ring-expanded product to eventually produce cephamycin C | Streptomyces clavuligerus ATCC 27064 | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
? | |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
assay at | Streptomyces clavuligerus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.054 | - |
penicillin G | recombinant wild-type enzyme, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 0.057 | - |
penicillin G | recombinant mutant T213V, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 0.064 | - |
penicillin G | recombinant mutant S261A, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 0.081 | - |
penicillin G | recombinant mutant Y184A, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 0.225 | - |
penicillin G | recombinant mutant Q126M, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 0.433 | - |
penicillin G | recombinant mutant S261M, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.0038 | - |
penicillin G | recombinant wild-type enzyme, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 0.0055 | - |
penicillin G | recombinant mutant Q126M, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 0.0058 | - |
penicillin G | recombinant mutant T213V, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 0.009 | - |
penicillin G | recombinant mutant S261M, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 0.0119 | - |
penicillin G | recombinant mutant S261A, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
| 0.0171 | - |
penicillin G | recombinant mutant Y184A, pH not specified in the publication, 30°C | Streptomyces clavuligerus | |
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