Cloned (Comment) | Organism |
---|---|
expression of His-tagged wild-type and mutant enzymes in Escherichia coli, expression of wild-type and mutants in Saccharomyces cerevisiae expressing also the CHS gene | Solanum lycopersicum |
Protein Variants | Comment | Organism |
---|---|---|
F239A | site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type | Solanum lycopersicum |
F239N | site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type | Solanum lycopersicum |
F239R | site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type | Solanum lycopersicum |
F239S | site-directed mutagenesis, the mutant shows increased activity in vivo, and in vitro with coumaric acid, but reduced activity with ferulic acid compared to the wild-type enzyme. The mutant strain produces more naringenin chalcone compared to the wild-type | Solanum lycopersicum |
F269G | site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type | Solanum lycopersicum |
F269I | site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type | Solanum lycopersicum |
F269L | site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type, loss of feedback inhibition by naringenin | Solanum lycopersicum |
F269L/K415T | site-directed mutagenesis, the mutant shows increased activity in vivo, and in vitro with coumaric acid, but reduced activity with ferulic acid compared to the wild-type enzyme | Solanum lycopersicum |
F269V | site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type | Solanum lycopersicum |
additional information | interactions of the Q274H mutation with other mutants, overview | Solanum lycopersicum |
Q274D | site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type | Solanum lycopersicum |
Q274D | site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type | Solanum lycopersicum |
Q274E | site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type | Solanum lycopersicum |
Q274G | site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type | Solanum lycopersicum |
Q274H | site-directed mutagenesis, the mutant shows increased activity in vivo, and in vitro with coumaric acid, but reduced activity with ferulic acid compared to the wild-type enzyme, loss of feedback inhibition by naringenin, the mutant strain produces more naringenin chalcone compared to the wild-type | Solanum lycopersicum |
Q274S | site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type | Solanum lycopersicum |
V186A | site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type | Solanum lycopersicum |
V186G | site-directed mutagenesis, the mutant shows highly increased activity in vivo, and in vitro with coumaric acid, but reduced activity with ferulic acid compared to the wild-type enzyme. The mutant strain produces highly increased naringenin chalcone content compared to the wild-type | Solanum lycopersicum |
V186I | site-directed mutagenesis, the mutant strain produces highly increased naringenin chalcone content compared to the wild-type | Solanum lycopersicum |
V186I/V187L | site-directed mutagenesis, the mutant shows increased activity in vivo, and in vitro with coumaric acid compared to the wild-type enzyme, but no activity with ferulic acid | Solanum lycopersicum |
V186L | site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type | Solanum lycopersicum |
V235M/A325G | site-directed mutagenesis, highly unstable inactive mutant | Solanum lycopersicum |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
naringenin | the wild-type enzyme is strongly allosterically inhibited by naringenin, a downstream product of the pathway. The feedback inhibition is probably exerted through a unique allosteric domain, docking-based model and in silico modeling for the interaction of naringenin with the wild-type enzyme. Mutations Q274H and F269L, but not V186G, lead to loss of feedback inhibition by naringenin | Solanum lycopersicum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | catalytic reaction scheme and kinetics, overview | Solanum lycopersicum | |
0.0036 | - |
4-coumarate | recombinant F269L/K415T, pH 7.8, 30°C | Solanum lycopersicum | |
0.0049 | - |
4-coumarate | recombinant V186I/V187L, pH 7.8, 30°C | Solanum lycopersicum | |
0.0051 | - |
4-coumarate | recombinant V186G, pH 7.8, 30°C | Solanum lycopersicum | |
0.00609 | - |
ferulate | recombinant wild-type enzyme, pH 7.8, 30°C | Solanum lycopersicum | |
0.00747 | - |
4-coumarate | recombinant wild-type enzyme, pH 7.8, 30°C | Solanum lycopersicum | |
0.0104 | - |
4-coumarate | recombinant F239S, pH 7.8, 30°C | Solanum lycopersicum | |
0.0106 | - |
ferulate | recombinant F269L/K415T, pH 7.8, 30°C | Solanum lycopersicum | |
0.0119 | - |
ferulate | recombinant F239S, pH 7.8, 30°C | Solanum lycopersicum | |
0.0142 | - |
ferulate | recombinant V186G, pH 7.8, 30°C | Solanum lycopersicum | |
0.0179 | - |
4-coumarate | recombinant Q274H, pH 7.8, 30°C | Solanum lycopersicum | |
0.0353 | - |
ferulate | recombinant Q274H, pH 7.8, 30°C | Solanum lycopersicum |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Solanum lycopersicum |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + 4-coumarate + CoA | Solanum lycopersicum | - |
AMP + diphosphate + 4-coumaroyl-CoA | - |
? | |
ATP + ferulate + CoA | Solanum lycopersicum | - |
AMP + diphosphate + ferulyl-CoA | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Solanum lycopersicum | - |
- |
- |
Purification (Comment) | Organism |
---|---|
expression of His-tagged wild-type and mutant enzymes in Escherichia coli | Solanum lycopersicum |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
ATP + 4-coumarate + CoA = AMP + diphosphate + 4-coumaroyl-CoA | bi uni uni bi ping pong mechanism, catalytic reaction scheme and kinetics, overview | Solanum lycopersicum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + 4-coumarate + CoA | - |
Solanum lycopersicum | AMP + diphosphate + 4-coumaroyl-CoA | - |
? | |
ATP + ferulate + CoA | - |
Solanum lycopersicum | AMP + diphosphate + ferulyl-CoA | - |
? |
Subunits | Comment | Organism |
---|---|---|
More | homology modeling | Solanum lycopersicum |
Synonyms | Comment | Organism |
---|---|---|
4-coumarate:CoA ligase | - |
Solanum lycopersicum |
4CL | - |
Solanum lycopersicum |
type I 4CL | - |
Solanum lycopersicum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Solanum lycopersicum |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.49 | - |
4-coumarate | recombinant V186I/V187L, pH 7.8, 30°C | Solanum lycopersicum | |
0.569 | - |
ferulate | recombinant V186G, pH 7.8, 30°C | Solanum lycopersicum | |
0.636 | - |
ferulate | recombinant F269L/K415T, pH 7.8, 30°C | Solanum lycopersicum | |
0.66 | - |
4-coumarate | recombinant wild-type enzyme, pH 7.8, 30°C | Solanum lycopersicum | |
0.742 | - |
4-coumarate | recombinant F269L/K415T, pH 7.8, 30°C | Solanum lycopersicum | |
1.063 | - |
ferulate | recombinant wild-type enzyme, pH 7.8, 30°C | Solanum lycopersicum | |
1.107 | - |
ferulate | recombinant F239S, pH 7.8, 30°C | Solanum lycopersicum | |
1.623 | - |
4-coumarate | recombinant V186G, pH 7.8, 30°C | Solanum lycopersicum | |
1.819 | - |
4-coumarate | recombinant F239S, pH 7.8, 30°C | Solanum lycopersicum | |
2.04 | - |
ferulate | recombinant Q274H, pH 7.8, 30°C | Solanum lycopersicum | |
4.05 | - |
4-coumarate | recombinant Q274H, pH 7.8, 30°C | Solanum lycopersicum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.8 | - |
assay at | Solanum lycopersicum |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Solanum lycopersicum |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme is a type I 4CL, phylogenetic analysis | Solanum lycopersicum |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
18.23 | - |
4-coumarate | recombinant V186G, pH 7.8, 30°C | Solanum lycopersicum | |
40.16 | - |
ferulate | recombinant V186G, pH 7.8, 30°C | Solanum lycopersicum | |
57.79 | - |
ferulate | recombinant Q274H, pH 7.8, 30°C | Solanum lycopersicum | |
60.06 | - |
ferulate | recombinant F269L/K415T, pH 7.8, 30°C | Solanum lycopersicum | |
88.4 | - |
4-coumarate | recombinant wild-type enzyme, pH 7.8, 30°C | Solanum lycopersicum | |
92.87 | - |
ferulate | recombinant F239S, pH 7.8, 30°C | Solanum lycopersicum | |
100 | - |
4-coumarate | recombinant V186I/V187L, pH 7.8, 30°C | Solanum lycopersicum | |
174.5 | - |
ferulate | recombinant wild-type enzyme, pH 7.8, 30°C | Solanum lycopersicum | |
174.6 | - |
4-coumarate | recombinant F239S, pH 7.8, 30°C | Solanum lycopersicum | |
206.1 | - |
4-coumarate | recombinant F269L/K415T, pH 7.8, 30°C | Solanum lycopersicum | |
226.3 | - |
4-coumarate | recombinant Q274H, pH 7.8, 30°C | Solanum lycopersicum |