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Literature summary extracted from

  • Alberstein, M.; Eisenstein, M.; Abeliovich, H.
    Removing allosteric feedback inhibition of tomato 4-coumarate:CoA ligase by directed evolution (2012), Plant J., 69, 57-69.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.2.1.12 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

EC Number Protein Variants Comment Organism
6.2.1.12 F239A site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 F239N site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 F239R site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 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
6.2.1.12 F269G site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 F269I site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 F269L site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type, loss of feedback inhibition by naringenin Solanum lycopersicum
6.2.1.12 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
6.2.1.12 F269V site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 additional information interactions of the Q274H mutation with other mutants, overview Solanum lycopersicum
6.2.1.12 Q274D site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 Q274D site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 Q274E site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 Q274G site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 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
6.2.1.12 Q274S site-directed mutagenesis, the mutant strain produces less naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 V186A site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 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
6.2.1.12 V186I site-directed mutagenesis, the mutant strain produces highly increased naringenin chalcone content compared to the wild-type Solanum lycopersicum
6.2.1.12 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
6.2.1.12 V186L site-directed mutagenesis, the mutant strain produces more naringenin chalcone compared to the wild-type Solanum lycopersicum
6.2.1.12 V235M/A325G site-directed mutagenesis, highly unstable inactive mutant Solanum lycopersicum

Inhibitors

EC Number Inhibitors Comment Organism Structure
6.2.1.12 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.2.1.12 additional information
-
 additional information catalytic reaction scheme and kinetics, overview Solanum lycopersicum
6.2.1.12 0.0036
-
 4-coumarate recombinant F269L/K415T, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.0049
-
 4-coumarate recombinant V186I/V187L, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.0051
-
 4-coumarate recombinant V186G, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.00609
-
 ferulate recombinant wild-type enzyme, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.00747
-
 4-coumarate recombinant wild-type enzyme, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.0104
-
 4-coumarate recombinant F239S, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.0106
-
 ferulate recombinant F269L/K415T, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.0119
-
 ferulate recombinant F239S, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.0142
-
 ferulate recombinant V186G, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.0179
-
 4-coumarate recombinant Q274H, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.0353
-
 ferulate recombinant Q274H, pH 7.8, 30°C Solanum lycopersicum

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.2.1.12 Mg2+ required Solanum lycopersicum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.2.1.12 ATP + 4-coumarate + CoA Solanum lycopersicum
-
 AMP + diphosphate + 4-coumaroyl-CoA
-
 ?
6.2.1.12 ATP + ferulate + CoA Solanum lycopersicum
-
 AMP + diphosphate + ferulyl-CoA
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.2.1.12 Solanum lycopersicum
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.2.1.12 expression of His-tagged wild-type and mutant enzymes in Escherichia coli Solanum lycopersicum

Reaction

EC Number Reaction Comment Organism Reaction ID
6.2.1.12 ATP + 4-coumarate + CoA = AMP + diphosphate + 4-coumaroyl-CoA bi uni uni bi ping pong mechanism, catalytic reaction scheme and kinetics, overview Solanum lycopersicum
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.2.1.12 ATP + 4-coumarate + CoA
-
 Solanum lycopersicum AMP + diphosphate + 4-coumaroyl-CoA
-
 ?
6.2.1.12 ATP + ferulate + CoA
-
 Solanum lycopersicum AMP + diphosphate + ferulyl-CoA
-
 ?

Subunits

EC Number Subunits Comment Organism
6.2.1.12 More homology modeling Solanum lycopersicum

Synonyms

EC Number Synonyms Comment Organism
6.2.1.12 4-coumarate:CoA ligase
-
 Solanum lycopersicum
6.2.1.12 4CL
-
 Solanum lycopersicum
6.2.1.12 type I 4CL
-
 Solanum lycopersicum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.2.1.12 30
-
 assay at Solanum lycopersicum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.2.1.12 0.49
-
 4-coumarate recombinant V186I/V187L, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.569
-
 ferulate recombinant V186G, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.636
-
 ferulate recombinant F269L/K415T, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.66
-
 4-coumarate recombinant wild-type enzyme, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 0.742
-
 4-coumarate recombinant F269L/K415T, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 1.063
-
 ferulate recombinant wild-type enzyme, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 1.107
-
 ferulate recombinant F239S, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 1.623
-
 4-coumarate recombinant V186G, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 1.819
-
 4-coumarate recombinant F239S, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 2.04
-
 ferulate recombinant Q274H, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 4.05
-
 4-coumarate recombinant Q274H, pH 7.8, 30°C Solanum lycopersicum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.2.1.12 7.8
-
 assay at Solanum lycopersicum

Cofactor

EC Number Cofactor Comment Organism Structure
6.2.1.12 ATP
-
 Solanum lycopersicum

General Information

EC Number General Information Comment Organism
6.2.1.12 evolution the enzyme is a type I 4CL, phylogenetic analysis Solanum lycopersicum

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
6.2.1.12 18.23
-
 4-coumarate recombinant V186G, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 40.16
-
 ferulate recombinant V186G, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 57.79
-
 ferulate recombinant Q274H, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 60.06
-
 ferulate recombinant F269L/K415T, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 88.4
-
 4-coumarate recombinant wild-type enzyme, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 92.87
-
 ferulate recombinant F239S, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 100
-
 4-coumarate recombinant V186I/V187L, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 174.5
-
 ferulate recombinant wild-type enzyme, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 174.6
-
 4-coumarate recombinant F239S, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 206.1
-
 4-coumarate recombinant F269L/K415T, pH 7.8, 30°C Solanum lycopersicum
6.2.1.12 226.3
-
 4-coumarate recombinant Q274H, pH 7.8, 30°C Solanum lycopersicum