Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in strain XL-1 Blue | Acremonium chrysogenum |
Protein Variants | Comment | Organism |
---|---|---|
DELTA310/M306I | site-directed mutagenesis, mutant selectively catalyzed the ring expansion with kinetics similar to the wild-type enzyme | Acremonium chrysogenum |
DELTA310/N305L/M306I | site-directed mutagenesis, mutant selectively catalyzed the ring expansion, and shows a lower Km value than the wild-type enzyme and about 3fold improved kinetic parameters | Acremonium chrysogenum |
M306I | site-directed mutagenesis, mutant shows no hydroxylation of desacetylcephalosporin C | Acremonium chrysogenum |
M306I | hydroxylation reaction of deacetoxycephalosporin C, E C1.14.11.26, is abolished, 59% of wild-type ring expansion activity | Acremonium chrysogenum |
additional information | DELTA310 is a C-terminally truncated mutant lacking residues 1-309, the mutant shows 2fold enhanced ring expansion activity with the artificial substrate penicillin G | Acremonium chrysogenum |
additional information | truncation of C-terminus to residue 310, 2fold enhancement of ring expansion reaction of penicillin G. Double mutant with truncation at residue 310 and M306I, selective catalyzation of ring expansion. Triple mutant with truncation at residue 310, M306I and N305L, selective catalization of ring expansion with improved kinetic parameters | Acremonium chrysogenum |
N305L | 107% of wild-type ring expansion activity, 85% of wild-type hydroxylation activity | Acremonium chrysogenum |
W82A | site-directed mutagenesis, mutant shows reduced ring expansion activity with artificial substrate penicillin G | Acremonium chrysogenum |
W82A | 5.5% of wild-type ring expansion activity, 71% of wild-type hydroxylation activity | Acremonium chrysogenum |
W82A | ring expansion reaction of EC 1.14.20.1 is reduced | Acremonium chrysogenum |
W82S | 44% of wild-type ring expansion activity, 18% of wild-type hydroxylation activity | Acremonium chrysogenum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2 | - |
penicillin G | pH 7.5, 30°C, recombinant mutant DELTA310/N305L/M306I | Acremonium chrysogenum | |
2 | - |
penicillin G | 30°C, triple mutant with truncation at residue 310, M306I and N305L | Acremonium chrysogenum | |
2.41 | - |
penicillin G | 30°C, mutant N305L | Acremonium chrysogenum | |
4.79 | - |
penicillin G | 30°C, mutant M306I | Acremonium chrysogenum | |
6.02 | - |
penicillin G | pH 7.5, 30°C, recombinant wild-type enzyme | Acremonium chrysogenum | |
6.04 | - |
penicillin G | 30°C, wild-type | Acremonium chrysogenum | |
6.42 | - |
penicillin G | 30°C, double mutant with truncation at residue 310 and M306I | Acremonium chrysogenum |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | dependent on, iron cofactor is bound by D184, H186, and H244 | Acremonium chrysogenum |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
36348 | - |
x * 36348, wild-type enzyme, electrospray mass spectrometry | Acremonium chrysogenum |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
deacetoxycephalosporin C + 2-oxoglutarate + O2 | Acremonium chrysogenum | - |
deacetylcephalosporin C + succinate + CO2 | - |
ir | |
penicillin N + 2-oxoglutarate + O2 | Acremonium chrysogenum | - |
deacetoxycephalosporin C + succinate + CO2 + H2O | - |
ir |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Acremonium chrysogenum | - |
catalyzes both reaction of EC 1.14.20.1 and of EC 1.14.11.26 | - |
Acremonium chrysogenum | P11935 | bifunctional enzyme | - |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
penicillin N + 2-oxoglutarate + O2 = deacetoxycephalosporin C + succinate + CO2 + H2O | bifunctional enzyme, residues W82, N305 and M306 are involved in catalysis, overview | Acremonium chrysogenum |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
activities of wild-type enzyme in ring-expansion and hydroxylation, cosubstrate and substrate conversion rate | Acremonium chrysogenum |
0.156 | - |
purified recombinant wild-type enzyme, substrate 6-alpha-methylpenicillin N, substrate conversion | Acremonium chrysogenum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
6-alpha-methylpenicillin N + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
? | |
6-alpha-methylpenicillin N + 2-oxoglutarate + O2 | best substrate in the ring expansion reaction | Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
ir | |
6-aminopenicillanic acid + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
? | |
6-aminopenicillanic acid + 2-oxoglutarate + O2 | low activity, ring expansion reaction | Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
ir | |
7-aminodeacetoxycephalosporanic acid + 2-oxoglutarate + O2 | low activity, hydroxylation reaction | Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
ir | |
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
? | |
acetyl-6-aminopenicillanic acid + 2-oxoglutarate + O2 | ring expansion reaction | Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
ir | |
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
? | |
adipyl-6-aminopenicillanic acid + 2-oxoglutarate + O2 | ring expansion reaction | Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
ir | |
amoxicillin + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
? | |
amoxicillin + 2-oxoglutarate + O2 | low activity, ring expansion reaction | Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
ir | |
ampicillin + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
? | |
ampicillin + 2-oxoglutarate + O2 | ring expansion reaction | Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
ir | |
cephalexin + 2-oxoglutarate + O2 | low activity, hydroxylation reaction | Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
ir | |
deacetoxycephalosporin C + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | deacetylcephalosporin C + succinate + CO2 | - |
ir | |
deacetoxycephalosporin C + 2-oxoglutarate + O2 | the penicillin /cephem substrate are bound by residues R161 and R163 | Acremonium chrysogenum | deacetylcephalosporin C + succinate + CO2 | - |
ir | |
additional information | deacetoxycephem substrate specificity for the hydroxylation reaction, penam substrate specificity of the bifunctional enzyme for the ring-expansion reation, overview | Acremonium chrysogenum | ? | - |
? | |
penicillin G + 2-oxoglutarate + O2 | the penicillin /cephem substrates are bound by residues R161 and R163 | Acremonium chrysogenum | phenylacetyl-7-aminodeacetoxycephalosporanic acid + succinate + CO2 + H2O | - |
ir | |
penicillin G + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
? | |
penicillin N + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
? | |
penicillin N + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
ir | |
penicillin N + 2-oxoglutarate + O2 | 2-oxoglutarate is required, residues M181, R259, and S261 might be responsible for binding of 2-oxoglutarate, the penicillin /cephem substrate are bound by residues R161 and R163 | Acremonium chrysogenum | deacetoxycephalosporin C + succinate + CO2 + H2O | - |
ir | |
penicillin V + 2-oxoglutarate + O2 | - |
Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
? | |
penicillin V + 2-oxoglutarate + O2 | low activity, ring expansion reaction | Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
ir | |
phenyl-7-aminodeacetoxycephalosporanic acid + 2-oxoglutarate + O2 | hydroxylation reaction | Acremonium chrysogenum | ? + succinate + CO2 + H2O | - |
ir |
Subunits | Comment | Organism |
---|---|---|
? | x * 36348, wild-type enzyme, electrospray mass spectrometry | Acremonium chrysogenum |
More | structural model of enzyme | Acremonium chrysogenum |
More | MW of mutant enzyme determined by electrospray mass spectrometry | Acremonium chrysogenum |
Synonyms | Comment | Organism |
---|---|---|
DAOC/DACS | - |
Acremonium chrysogenum |
deacetoxycephalosporin/deacetylcephalosporin C synthase | - |
Acremonium chrysogenum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Acremonium chrysogenum |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.016 | - |
penicillin G | 30°C, mutant M306I | Acremonium chrysogenum | |
0.07 | - |
penicillin G | 30°C, triple mutant with truncation at residue 310, M306I and N305L | Acremonium chrysogenum | |
0.073 | - |
penicillin G | pH 7.5, 30°C, recombinant wild-type enzyme | Acremonium chrysogenum | |
0.073 | - |
penicillin G | 30°C, wild-type | Acremonium chrysogenum | |
0.079 | - |
penicillin G | 30°C, mutant N305L | Acremonium chrysogenum | |
0.091 | - |
penicillin G | 30°C, double mutant with truncation at residue 310 and M306I | Acremonium chrysogenum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Acremonium chrysogenum |