Literature summary for 6.2.1.12 extracted from

Li, Z.; Nair, S.K.
Structural basis for specificity and flexibility in a plant 4-coumarate CoA ligase (2015), Structure, 23, 2032-2042 .

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli Rosetta (DE3) cells	Nicotiana tabacum

Crystallization (Commentary)

Crystallization (Comment)	Organism
isoform 4CL2 in complex with Mg2+ and ATP or with AMP and coenzyme A or with three different hydroxycinnamate-AMP intermediates: 4-coumaroyl-AMP, caffeoyl-AMP, and feruloyl-AMP, hanging drop vapor diffusion method, using 20% (w/v) PEG 3350 and 0.2 M potassium nitrate or 4.4% (w/v) PEG 8000, 0.08 M sodium cacodylate (pH 6.5), 0.16 M calcium acetate, and 20% (v/v) glycerol or 25.5% (w/v) PEG 8000, 0.085 M sodium cacodylate (pH 6.5), 0.17 M sodium acetate, and 15% (v/v) glycerol or 25% (w/v) PEG 3350, 0.2 M ammonium acetate, and 0.1 M Tris (pH 8.5)	Nicotiana tabacum

Crystallization (Comment)

Organism

isoform 4CL2 in complex with Mg2+ and ATP or with AMP and coenzyme A or with three different hydroxycinnamate-AMP intermediates: 4-coumaroyl-AMP, caffeoyl-AMP, and feruloyl-AMP, hanging drop vapor diffusion method, using 20% (w/v) PEG 3350 and 0.2 M potassium nitrate or 4.4% (w/v) PEG 8000, 0.08 M sodium cacodylate (pH 6.5), 0.16 M calcium acetate, and 20% (v/v) glycerol or 25.5% (w/v) PEG 8000, 0.085 M sodium cacodylate (pH 6.5), 0.17 M sodium acetate, and 15% (v/v) glycerol or 25% (w/v) PEG 3350, 0.2 M ammonium acetate, and 0.1 M Tris (pH 8.5)

Nicotiana tabacum

Protein Variants

Protein Variants	Comment	Organism
H237A	the mutant shows reduced activity compared to the wild type enzyme	Nicotiana tabacum
K197A	the mutant shows reduced activity compared to the wild type enzyme	Nicotiana tabacum
K441A	inactive	Nicotiana tabacum
K443A	the mutant shows reduced activity compared to the wild type enzyme	Nicotiana tabacum
K526A	inactive	Nicotiana tabacum
M344A	the mutant shows reduced activity compared to the wild type enzyme	Nicotiana tabacum
R435A	the mutant shows reduced activity compared to the wild type enzyme	Nicotiana tabacum
T193A	the mutant shows reduced activity compared to the wild type enzyme	Nicotiana tabacum
T336A	the mutant shows reduced activity compared to the wild type enzyme	Nicotiana tabacum
Y239A	the mutant shows reduced activity compared to the wild type enzyme	Nicotiana tabacum
Y239F	the mutant shows reduced activity compared to the wild type enzyme	Nicotiana tabacum

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.0009	-	4-coumarate	mutant enzyme K443A, at pH 7.5 and 25°C	Nicotiana tabacum
0.001	-	caffeate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum
0.0015	-	4-coumarate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum
0.0029	-	ferulate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum
0.0029	-	4-coumarate	mutant enzyme T193A, at pH 7.5 and 25°C	Nicotiana tabacum
0.0051	-	4-coumarate	mutant enzyme T336A, at pH 7.5 and 25°C	Nicotiana tabacum
0.0069	-	4-coumarate	mutant enzyme Y239F, at pH 7.5 and 25°C	Nicotiana tabacum
0.0139	-	4-coumarate	mutant enzyme K197A, at pH 7.5 and 25°C	Nicotiana tabacum
0.1434	-	4-coumarate	mutant enzyme H237A, at pH 7.5 and 25°C	Nicotiana tabacum
0.2281	-	4-coumarate	mutant enzyme Y239A, at pH 7.5 and 25°C	Nicotiana tabacum
0.5207	-	4-coumarate	mutant enzyme M344A, at pH 7.5 and 25°C	Nicotiana tabacum
0.6149	-	4-coumarate	mutant enzyme R435A, at pH 7.5 and 25°C	Nicotiana tabacum
1.32	-	sinapinate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Nicotiana tabacum

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + 4-coumarate + CoA	Nicotiana tabacum	-	AMP + diphosphate + 4-coumaroyl-CoA	-	?

Organism

Organism	UniProt	Comment	Textmining
Nicotiana tabacum	O24146	-	-

Purification (Commentary)

Purification (Comment)	Organism
metal ion column chromatography and gel filtration	Nicotiana tabacum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + 4-coumarate + CoA	-	Nicotiana tabacum	AMP + diphosphate + 4-coumaroyl-CoA	-	?
ATP + caffeate + CoA	-	Nicotiana tabacum	AMP + diphosphate + caffeoyl-CoA	-	?
ATP + ferulate + CoA	-	Nicotiana tabacum	AMP + diphosphate + feruloyl-CoA	-	?
ATP + sinapinate + CoA	-	Nicotiana tabacum	AMP + diphosphate + sinapoyl-CoA	-	?

Synonyms

Synonyms	Comment	Organism
4-coumarate:CoA ligase	-	Nicotiana tabacum
4CL2	isoform	Nicotiana tabacum

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.018	-	sinapinate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum
0.282	-	4-coumarate	mutant enzyme T336A, at pH 7.5 and 25°C	Nicotiana tabacum
0.996	-	4-coumarate	mutant enzyme T193A, at pH 7.5 and 25°C	Nicotiana tabacum
0.996	-	4-coumarate	mutant enzyme Y239A, at pH 7.5 and 25°C	Nicotiana tabacum
1.47	-	4-coumarate	mutant enzyme K443A, at pH 7.5 and 25°C	Nicotiana tabacum
1.98	-	4-coumarate	mutant enzyme H237A, at pH 7.5 and 25°C	Nicotiana tabacum
2.652	-	caffeate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum
3.036	-	4-coumarate	mutant enzyme R435A, at pH 7.5 and 25°C	Nicotiana tabacum
3.576	-	ferulate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum
3.978	-	4-coumarate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum
4.47	-	4-coumarate	mutant enzyme Y239F, at pH 7.5 and 25°C	Nicotiana tabacum
7.11	-	4-coumarate	mutant enzyme K197A, at pH 7.5 and 25°C	Nicotiana tabacum
9.144	-	4-coumarate	mutant enzyme M344A, at pH 7.5 and 25°C	Nicotiana tabacum

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Nicotiana tabacum

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.0136	-	sinapinate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum
4.367	-	4-coumarate	mutant enzyme Y239A, at pH 7.5 and 25°C	Nicotiana tabacum
4.937	-	4-coumarate	mutant enzyme R435A, at pH 7.5 and 25°C	Nicotiana tabacum
13.81	-	4-coumarate	mutant enzyme H237A, at pH 7.5 and 25°C	Nicotiana tabacum
55.29	-	4-coumarate	mutant enzyme T336A, at pH 7.5 and 25°C	Nicotiana tabacum
175.6	-	4-coumarate	mutant enzyme M344A, at pH 7.5 and 25°C	Nicotiana tabacum
343.4	-	4-coumarate	mutant enzyme T193A, at pH 7.5 and 25°C	Nicotiana tabacum
511.5	-	4-coumarate	mutant enzyme K197A, at pH 7.5 and 25°C	Nicotiana tabacum
647.8	-	4-coumarate	mutant enzyme Y239F, at pH 7.5 and 25°C	Nicotiana tabacum
1233	-	ferulate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum
1633	-	4-coumarate	mutant enzyme K443A, at pH 7.5 and 25°C	Nicotiana tabacum
2652	-	4-coumarate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum
2652	-	caffeate	wild type enzyme, at pH 7.5 and 25°C	Nicotiana tabacum