EC Number | Activating Compound | Comment | Organism | Structure |
---|---|---|---|---|
1.14.11.26 | ascorbate | required | Streptomyces clavuligerus |
EC Number | Cloned (Comment) | Organism |
---|---|---|
1.14.11.26 | geen cefF, recombinant exression of cefF library mutants, site-directed mutants, and wild-type enzyme in Escherichia coli strain BL21(DE3) | Streptomyces clavuligerus |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
1.14.11.26 | A177V | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | A311V | random mutagenesis | Streptomyces clavuligerus |
1.14.11.26 | E16G/T90A/T304A | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | E16G/T90A/T304A | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | E209Q | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | E82D | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | F267L | random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | I193V | random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | L236V | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | M184I | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | M229V | random mutagenesis | Streptomyces clavuligerus |
1.14.11.26 | additional information | mutagenic cefF library creation by random mutagenesis and screening of the mutant deacetylcephalosporin C synthase enzyme library, homology modeling of DACS structure and mapping of mutant positions. Process-level biotransformation reaction of cephalosporin G to deacetylcephalosporin G by mutants of deacetylcephalosporin C synthase. Deacetylcephalosporin G can be converted completely into hydroxymethyl-7-amino-cephalosporanic acid (HACA) in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. Directed-evolution strategies such as random, semirational, rational, and computational methods are used for systematic engineering of DACS for improved activity with cephalosporin G | Streptomyces clavuligerus |
1.14.11.26 | P186L | random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | P72L | random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | R182S | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | R182W | random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | S251F | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | S260G | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T273A | random mutagenesis | Streptomyces clavuligerus |
1.14.11.26 | T90A | random mutagenesis | Streptomyces clavuligerus |
1.14.11.26 | T90A/A311V | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/M184I/I193V/F267L | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/R182S | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/S251F | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/S260G | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V171L/F267L | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/A241V/V307A | site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D | site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/N313D | site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/R91G | site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/R91G/A241V/V307A | site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/T96S/A241V/V307A | site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/T96S/G255D/A280S | site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/V226I | site-directed mutagenesis, the mutant shows highly increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/A40V/M229I/T273A/V221P | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/F195L | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/P7L/A237V | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/P7L/T273A | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/R250L | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/V206I/A210V | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | T90A/P72L/A311V/V206I/A210V/T273A | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | V150A | random mutagenesis | Streptomyces clavuligerus |
1.14.11.26 | V171L | random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | V171M | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | V221A | random mutagenesis | Streptomyces clavuligerus |
1.14.11.26 | V221H | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | V221P | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | V221T | random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | V249I | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
1.14.11.26 | Y38C | random mutagenesis | Streptomyces clavuligerus |
EC Number | KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.14.11.26 | 2.58 | - |
cephalosporin G | pH 7.5, 25°C, recombinant wild-type enzyme | Streptomyces clavuligerus | |
1.14.11.26 | 2.58 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V | Streptomyces clavuligerus | |
1.14.11.26 | 4.41 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D | Streptomyces clavuligerus | |
1.14.11.26 | 4.64 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A | Streptomyces clavuligerus | |
1.14.11.26 | 7.24 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus | |
1.14.11.26 | 9.2 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
1.14.11.26 | Fe2+ | dependent on, required for catalysis | Streptomyces clavuligerus |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.14.11.26 | deacetoxycephalosporin C + 2-oxoglutarate + O2 | Streptomyces clavuligerus | - |
deacetylcephalosporin C + succinate + CO2 | - |
? | |
1.14.11.26 | deacetoxycephalosporin C + 2-oxoglutarate + O2 | Streptomyces clavuligerus ATCC 27064 | - |
deacetylcephalosporin C + succinate + CO2 | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
1.14.11.26 | Streptomyces clavuligerus | P42220 | - |
- |
1.14.11.26 | Streptomyces clavuligerus ATCC 27064 | P42220 | - |
- |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
1.14.11.26 | cephalosporin G + 2-oxoglutarate + O2 | the wild-type enzyme shows negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, but diverse enzyme mutants show increased to high activity with cephalosporin G, overview | Streptomyces clavuligerus | deacetylcephalosporin G + succinate + CO2 | - |
? | |
1.14.11.26 | cephalosporin G + 2-oxoglutarate + O2 | the wild-type enzyme shows negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, but diverse enzyme mutants show increased to high activity with cephalosporin G, overview | Streptomyces clavuligerus ATCC 27064 | deacetylcephalosporin G + succinate + CO2 | - |
? | |
1.14.11.26 | deacetoxycephalosporin C + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus | deacetylcephalosporin C + succinate + CO2 | - |
? | |
1.14.11.26 | deacetoxycephalosporin C + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus ATCC 27064 | deacetylcephalosporin C + succinate + CO2 | - |
? |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
1.14.11.26 | cefF | - |
Streptomyces clavuligerus |
1.14.11.26 | DACS | - |
Streptomyces clavuligerus |
1.14.11.26 | deacetylcephalosporin C synthase | - |
Streptomyces clavuligerus |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
1.14.11.26 | 25 | - |
assay at | Streptomyces clavuligerus |
EC Number | Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.14.11.26 | 0.055 | - |
cephalosporin G | pH 7.5, 25°C, recombinant wild-type enzyme | Streptomyces clavuligerus | |
1.14.11.26 | 0.122 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V | Streptomyces clavuligerus | |
1.14.11.26 | 0.326 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A | Streptomyces clavuligerus | |
1.14.11.26 | 1.805 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus | |
1.14.11.26 | 1.899 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D | Streptomyces clavuligerus | |
1.14.11.26 | 2.302 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
1.14.11.26 | 7.5 | - |
assay at | Streptomyces clavuligerus |
EC Number | General Information | Comment | Organism |
---|---|---|---|
1.14.11.26 | evolution | the enzyme belongs to the family of 2-oxoglutarate-dependent oxygenases | Streptomyces clavuligerus |
1.14.11.26 | metabolism | deacetylcephalosporin C synthase (DACS) transforms an inert methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) during the biosynthesis of cephalosporins | Streptomyces clavuligerus |
1.14.11.26 | additional information | homology modeling of DACS structure | Streptomyces clavuligerus |
1.14.11.26 | physiological function | the enzyme is involved in the biosynthesis of cephalosporins, it carries out a critical transformation of an inert cephem methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) in the presence of iron, oxygen, ascorbic acid, and 2-oxoglutarate. It is a step which is chemically difficult to accomplish and which offers a second substitution site for derivatization in cephalosporins | Streptomyces clavuligerus |
EC Number | kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|---|
1.14.11.26 | 0.021 | - |
cephalosporin G | pH 7.5, 25°C, recombinant wild-type enzyme | Streptomyces clavuligerus | |
1.14.11.26 | 0.047 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V | Streptomyces clavuligerus | |
1.14.11.26 | 0.07 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A | Streptomyces clavuligerus | |
1.14.11.26 | 0.249 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus | |
1.14.11.26 | 0.25 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus | |
1.14.11.26 | 0.43 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D | Streptomyces clavuligerus |