Activating Compound | Comment | Organism | Structure |
---|---|---|---|
ascorbate | required | Streptomyces clavuligerus |
Cloned (Comment) | Organism |
---|---|
geen cefF, recombinant exression of cefF library mutants, site-directed mutants, and wild-type enzyme in Escherichia coli strain BL21(DE3) | Streptomyces clavuligerus |
Protein Variants | Comment | Organism |
---|---|---|
A177V | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
A311V | random mutagenesis | Streptomyces clavuligerus |
E16G/T90A/T304A | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
E16G/T90A/T304A | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
E209Q | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
E82D | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
F267L | random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
I193V | random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
L236V | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
M184I | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
M229V | random mutagenesis | Streptomyces clavuligerus |
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 |
P186L | random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
P72L | random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
R182S | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
R182W | random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
S251F | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
S260G | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
T273A | random mutagenesis | Streptomyces clavuligerus |
T90A | random mutagenesis | Streptomyces clavuligerus |
T90A/A311V | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
T90A/P72L | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
T90A/P72L/A311V | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
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 |
T90A/P72L/A311V/F195L | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
T90A/P72L/A311V/P7L/A237V | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
T90A/P72L/A311V/P7L/T273A | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
T90A/P72L/A311V/R250L | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
T90A/P72L/A311V/V206I/A210V | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
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 |
V150A | random mutagenesis | Streptomyces clavuligerus |
V171L | random mutagenesis, active site mutation, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
V171M | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
V221A | random mutagenesis | Streptomyces clavuligerus |
V221H | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
V221P | site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
V221T | random mutagenesis and site-directed mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
V249I | random mutagenesis, the mutant shows increased activity with cephalosporin G compared to the wild-type enzyme | Streptomyces clavuligerus |
Y38C | random mutagenesis | Streptomyces clavuligerus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.58 | - |
cephalosporin G | pH 7.5, 25°C, recombinant wild-type enzyme | Streptomyces clavuligerus | |
2.58 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V | Streptomyces clavuligerus | |
4.41 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D | Streptomyces clavuligerus | |
4.64 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A | Streptomyces clavuligerus | |
7.24 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus | |
9.2 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | dependent on, required for catalysis | Streptomyces clavuligerus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
deacetoxycephalosporin C + 2-oxoglutarate + O2 | Streptomyces clavuligerus | - |
deacetylcephalosporin C + succinate + CO2 | - |
? | |
deacetoxycephalosporin C + 2-oxoglutarate + O2 | Streptomyces clavuligerus ATCC 27064 | - |
deacetylcephalosporin C + succinate + CO2 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Streptomyces clavuligerus | P42220 | - |
- |
Streptomyces clavuligerus ATCC 27064 | P42220 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | - |
? | |
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 | - |
? | |
deacetoxycephalosporin C + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus | deacetylcephalosporin C + succinate + CO2 | - |
? | |
deacetoxycephalosporin C + 2-oxoglutarate + O2 | - |
Streptomyces clavuligerus ATCC 27064 | deacetylcephalosporin C + succinate + CO2 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
cefF | - |
Streptomyces clavuligerus |
DACS | - |
Streptomyces clavuligerus |
deacetylcephalosporin C synthase | - |
Streptomyces clavuligerus |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Streptomyces clavuligerus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.055 | - |
cephalosporin G | pH 7.5, 25°C, recombinant wild-type enzyme | Streptomyces clavuligerus | |
0.122 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V | Streptomyces clavuligerus | |
0.326 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A | Streptomyces clavuligerus | |
1.805 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus | |
1.899 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D | Streptomyces clavuligerus | |
2.302 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Streptomyces clavuligerus |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the family of 2-oxoglutarate-dependent oxygenases | Streptomyces clavuligerus |
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 |
additional information | homology modeling of DACS structure | Streptomyces clavuligerus |
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 |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.021 | - |
cephalosporin G | pH 7.5, 25°C, recombinant wild-type enzyme | Streptomyces clavuligerus | |
0.047 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V | Streptomyces clavuligerus | |
0.07 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A | Streptomyces clavuligerus | |
0.249 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus | |
0.25 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L | Streptomyces clavuligerus | |
0.43 | - |
cephalosporin G | pH 7.5, 25°C, recombinant mutant T90A/P72L/A311V/A40V/M229I/T273A/V171L/R182W/F267L/G108D | Streptomyces clavuligerus |