Cloned (Comment) | Organism |
---|---|
expression of the His-tagged wild-type and mutant enzymes in Escherichia coli JM109 | Corynebacterium glutamicum |
Protein Variants | Comment | Organism |
---|---|---|
E64D | constructed by PCR-based random mutagenesis and complementation analysis, 15% reduced activity compared to the wild-type enzyme, 4.5% increased Km, 1.7fold increased kcat | Corynebacterium glutamicum |
E64Q | constructed by PCR-based random mutagenesis and complementation analysis, inactive mutant | Corynebacterium glutamicum |
E64S | constructed by PCR-based random mutagenesis and complementation analysis, inactive mutant | Corynebacterium glutamicum |
E64V | constructed by PCR-based random mutagenesis and complementation analysis, inactive mutant | Corynebacterium glutamicum |
F185L | constructed by PCR-based random mutagenesis and complementation analysis, inactive mutant | Corynebacterium glutamicum |
F185Y | constructed by PCR-based random mutagenesis and complementation analysis, 96% reduced activity compared to the wild-type enzyme, 26% increased Km | Corynebacterium glutamicum |
additional information | alterations of Asp76, Glu89, His115, and Arg236 do not cause significant changes in the kinetics properties | Corynebacterium glutamicum |
R184L | constructed by PCR-based random mutagenesis and complementation analysis, 50% reduced activity compared to the wild-type enzyme | Corynebacterium glutamicum |
S99A | constructed by PCR-based random mutagenesis and complementation analysis, insensitive to inhibition by phenylalanine | Corynebacterium glutamicum |
S99C | constructed by PCR-based random mutagenesis and complementation analysis, insensitive to inhibition by phenylalanine | Corynebacterium glutamicum |
S99L | constructed by PCR-based random mutagenesis and complementation analysis, insensitive to inhibition by phenylalanine | Corynebacterium glutamicum |
S99M | constructed by PCR-based random mutagenesis and complementation analysis, 30% reduced activity compared to the wild-type enzyme, insensitive to inhibition by phenylalanine | Corynebacterium glutamicum |
T183A | constructed by PCR-based random mutagenesis and complementation analysis, inactive mutant | Corynebacterium glutamicum |
T183S | constructed by PCR-based random mutagenesis and complementation analysis, highly reduced activity | Corynebacterium glutamicum |
T183Y | constructed by PCR-based random mutagenesis and complementation analysis, inactive mutant | Corynebacterium glutamicum |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
L-phenylalanine | feedback inhibition, Ser99 is involved, mutants S99M, S99T, S99A, S99C, or S99L are not sensitive to inhibition | Corynebacterium glutamicum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.07 | - |
prephenate | wild-type enzyme, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.08 | - |
prephenate | mutant S99T, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.09 | - |
prephenate | mutant S99C, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.13 | - |
prephenate | mutant S99A, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.18 | - |
prephenate | mutant S99M, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.29 | - |
prephenate | mutant E164D, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.46 | - |
prephenate | mutant T183S, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.61 | - |
prephenate | mutant R184L, pH 7.5, 30°C | Corynebacterium glutamicum | |
1.73 | - |
prephenate | mutant F185Y, pH 7.5, 30°C | Corynebacterium glutamicum |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
39000 | - |
x * 39000, recombinant wild-type and mutant enzymes, SDS-PAGE | Corynebacterium glutamicum |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
prephenate | Corynebacterium glutamicum | enzyme is a key regulatory enzyme in the phenylalanine-specific pathway | phenylpyruvate + H2O + CO2 | - |
? | |
prephenate | Corynebacterium glutamicum CCRC 11384 | enzyme is a key regulatory enzyme in the phenylalanine-specific pathway | phenylpyruvate + H2O + CO2 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Corynebacterium glutamicum | - |
- |
- |
Corynebacterium glutamicum CCRC 11384 | - |
- |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli | Corynebacterium glutamicum |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
prephenate = phenylpyruvate + H2O + CO2 | this enzyme in the enteric bacteria also possesses chorismate mutase activity, EC 5.4.99.5, and converts chorismate into prephenate, residues Glu64, Thr183, Arg184, and Phe185 might be involved in catalysis and substrate binding | Corynebacterium glutamicum |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.32 | - |
mutant F185Y, pH 7.5, 30°C | Corynebacterium glutamicum |
0.39 | - |
mutant T183S, pH 7.5, 30°C | Corynebacterium glutamicum |
2.37 | - |
mutant S99A, pH 7.5, 30°C | Corynebacterium glutamicum |
4.31 | - |
mutant R184L, pH 7.5, 30°C | Corynebacterium glutamicum |
6.66 | - |
mutant S99M, pH 7.5, 30°C | Corynebacterium glutamicum |
6.88 | - |
mutant S99C, pH 7.5, 30°C | Corynebacterium glutamicum |
7.59 | - |
mutant S99T, pH 7.5, 30°C | Corynebacterium glutamicum |
8.1 | - |
mutant E164D, pH 7.5, 30°C | Corynebacterium glutamicum |
9.48 | - |
wild-type enzyme, pH 7.5, 30°C | Corynebacterium glutamicum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
prephenate | - |
Corynebacterium glutamicum | phenylpyruvate + H2O + CO2 | - |
? | |
prephenate | enzyme is a key regulatory enzyme in the phenylalanine-specific pathway | Corynebacterium glutamicum | phenylpyruvate + H2O + CO2 | - |
? | |
prephenate | - |
Corynebacterium glutamicum CCRC 11384 | phenylpyruvate + H2O + CO2 | - |
? | |
prephenate | enzyme is a key regulatory enzyme in the phenylalanine-specific pathway | Corynebacterium glutamicum CCRC 11384 | phenylpyruvate + H2O + CO2 | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 39000, recombinant wild-type and mutant enzymes, SDS-PAGE | Corynebacterium glutamicum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Corynebacterium glutamicum |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.01 | - |
prephenate | mutant F185Y, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.01 | - |
prephenate | mutant T183S, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.03 | - |
prephenate | mutant S99A, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.09 | - |
prephenate | mutant S99C, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.11 | - |
prephenate | mutant S99T, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.12 | - |
prephenate | mutant S99M, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.12 | - |
prephenate | wild-type enzyme, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.13 | - |
prephenate | mutant R184L, pH 7.5, 30°C | Corynebacterium glutamicum | |
0.21 | - |
prephenate | mutant E164D, pH 7.5, 30°C | Corynebacterium glutamicum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Corynebacterium glutamicum |