Catalytic mechanism of SHCHC synthase in the menaquinone biosynthesis of Escherichia coli: identification and mutational analysis of the active site residues
Jiang, M.; Chen, X.; Wu, X.H.; Chen, M.; Wu, Y.D.; Guo, Z.; Biochemistry 48, 6921-6931 (2009) 
Data extracted from this reference:
Cloned(Commentary)
EC Number
Cloned (Commentary)
Organism
4.2.99.20
expressed in Escherichia coli BL21(DE3)
Escherichia coli
Engineering
EC Number
Protein Variants
Commentary
Organism
4.2.99.20
D128A
conserved among MenH proteins
Escherichia coli
4.2.99.20
D210A
part of the catalytically essential triad
Escherichia coli
4.2.99.20
H232A
part of the catalytically essential triad
Escherichia coli
4.2.99.20
H232K
part of the catalytically essential triad
Escherichia coli
4.2.99.20
K212A
conserved among MenH proteins
Escherichia coli
4.2.99.20
R124A
conserved among MenH proteins
Escherichia coli
4.2.99.20
R168A
conserved among MenH proteins
Escherichia coli
4.2.99.20
R90A
conserved among MenH proteins
Escherichia coli
4.2.99.20
S86A
part of the catalytically essential triad
Escherichia coli
4.2.99.20
W147A
conserved among MenH proteins
Escherichia coli
4.2.99.20
W147F
conserved among MenH proteins
Escherichia coli
4.2.99.20
Y85F
conserved among MenH proteins
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.2.99.20
0.0086
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232A mutant protein
Escherichia coli
4.2.99.20
0.01
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
wild-type protein
Escherichia coli
4.2.99.20
0.014
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
Y85F mutant protein
Escherichia coli
4.2.99.20
0.0194
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
K212A mutant protein
Escherichia coli
4.2.99.20
0.0237
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147A mutant protein
Escherichia coli
4.2.99.20
0.028
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
S86A mutant protein
Escherichia coli
4.2.99.20
0.074
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147F mutant protein
Escherichia coli
4.2.99.20
0.084
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R124A mutant protein
Escherichia coli
4.2.99.20
0.085
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D128A mutant protein
Escherichia coli
4.2.99.20
0.118
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D210A mutant protein
Escherichia coli
4.2.99.20
0.134
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R168A mutant protein
Escherichia coli
4.2.99.20
0.159
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R90A mutant protein
Escherichia coli
4.2.99.20
0.532
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232K mutant protein
Escherichia coli
Organism
EC Number
Organism
UniProt
Commentary
Textmining
4.2.99.20
Escherichia coli
-
-
-
Purification (Commentary)
EC Number
Purification (Commentary)
Organism
4.2.99.20
immobilized metal ion affinity chromatography, gel filtration
Escherichia coli
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
Substrate Product ID
4.2.99.20
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
-
702326
Escherichia coli
(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate + pyruvate
-
-
-
?
Synonyms
EC Number
Synonyms
Commentary
Organism
4.2.99.20
(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase
-
Escherichia coli
4.2.99.20
MenH
-
Escherichia coli
4.2.99.20
SHCHC
-
Escherichia coli
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.2.99.20
0.00058
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232A mutant protein
Escherichia coli
4.2.99.20
0.021
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232K mutant protein
Escherichia coli
4.2.99.20
0.047
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R90A mutant protein
Escherichia coli
4.2.99.20
0.056
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147A mutant protein
Escherichia coli
4.2.99.20
0.18
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D210A mutant protein
Escherichia coli
4.2.99.20
0.29
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
S86A mutant protein
Escherichia coli
4.2.99.20
0.51
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R124A mutant protein
Escherichia coli
4.2.99.20
2.58
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
Y85F mutant protein
Escherichia coli
4.2.99.20
2.9
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R168A mutant protein
Escherichia coli
4.2.99.20
15.4
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147F mutant protein
Escherichia coli
4.2.99.20
41.5
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
K212A mutant protein
Escherichia coli
4.2.99.20
115
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D128A mutant protein
Escherichia coli
4.2.99.20
147
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
wild-type protein
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.2.99.20
expressed in Escherichia coli BL21(DE3)
Escherichia coli
Engineering (protein specific)
EC Number
Protein Variants
Commentary
Organism
4.2.99.20
D128A
conserved among MenH proteins
Escherichia coli
4.2.99.20
D210A
part of the catalytically essential triad
Escherichia coli
4.2.99.20
H232A
part of the catalytically essential triad
Escherichia coli
4.2.99.20
H232K
part of the catalytically essential triad
Escherichia coli
4.2.99.20
K212A
conserved among MenH proteins
Escherichia coli
4.2.99.20
R124A
conserved among MenH proteins
Escherichia coli
4.2.99.20
R168A
conserved among MenH proteins
Escherichia coli
4.2.99.20
R90A
conserved among MenH proteins
Escherichia coli
4.2.99.20
S86A
part of the catalytically essential triad
Escherichia coli
4.2.99.20
W147A
conserved among MenH proteins
Escherichia coli
4.2.99.20
W147F
conserved among MenH proteins
Escherichia coli
4.2.99.20
Y85F
conserved among MenH proteins
Escherichia coli
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.2.99.20
0.0086
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232A mutant protein
Escherichia coli
4.2.99.20
0.01
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
wild-type protein
Escherichia coli
4.2.99.20
0.014
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
Y85F mutant protein
Escherichia coli
4.2.99.20
0.0194
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
K212A mutant protein
Escherichia coli
4.2.99.20
0.0237
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147A mutant protein
Escherichia coli
4.2.99.20
0.028
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
S86A mutant protein
Escherichia coli
4.2.99.20
0.074
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147F mutant protein
Escherichia coli
4.2.99.20
0.084
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R124A mutant protein
Escherichia coli
4.2.99.20
0.085
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D128A mutant protein
Escherichia coli
4.2.99.20
0.118
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D210A mutant protein
Escherichia coli
4.2.99.20
0.134
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R168A mutant protein
Escherichia coli
4.2.99.20
0.159
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R90A mutant protein
Escherichia coli
4.2.99.20
0.532
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232K mutant protein
Escherichia coli
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.2.99.20
immobilized metal ion affinity chromatography, gel filtration
Escherichia coli
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ID
4.2.99.20
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
-
702326
Escherichia coli
(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate + pyruvate
-
-
-
?
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.2.99.20
0.00058
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232A mutant protein
Escherichia coli
4.2.99.20
0.021
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232K mutant protein
Escherichia coli
4.2.99.20
0.047
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R90A mutant protein
Escherichia coli
4.2.99.20
0.056
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147A mutant protein
Escherichia coli
4.2.99.20
0.18
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D210A mutant protein
Escherichia coli
4.2.99.20
0.29
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
S86A mutant protein
Escherichia coli
4.2.99.20
0.51
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R124A mutant protein
Escherichia coli
4.2.99.20
2.58
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
Y85F mutant protein
Escherichia coli
4.2.99.20
2.9
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R168A mutant protein
Escherichia coli
4.2.99.20
15.4
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147F mutant protein
Escherichia coli
4.2.99.20
41.5
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
K212A mutant protein
Escherichia coli
4.2.99.20
115
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D128A mutant protein
Escherichia coli
4.2.99.20
147
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
wild-type protein
Escherichia coli
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
4.2.99.20
0.0392
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232K mutant protein
Escherichia coli
4.2.99.20
0.067
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232A mutant protein
Escherichia coli
4.2.99.20
0.295
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R90A mutant protein
Escherichia coli
4.2.99.20
1.5
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D210A mutant protein
Escherichia coli
4.2.99.20
2.4
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147A mutant protein
Escherichia coli
4.2.99.20
6.1
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R124A mutant protein
Escherichia coli
4.2.99.20
10
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
S86A mutant protein
Escherichia coli
4.2.99.20
15
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R168A mutant protein
Escherichia coli
4.2.99.20
180
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
Y85F mutant protein
Escherichia coli
4.2.99.20
220
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147F mutant protein
Escherichia coli
4.2.99.20
1400
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D128A mutant protein
Escherichia coli
4.2.99.20
2200
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
K212A mutant protein
Escherichia coli
4.2.99.20
14000
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
wild-type protein
Escherichia coli
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
4.2.99.20
0.0392
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232K mutant protein
Escherichia coli
4.2.99.20
0.067
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
H232A mutant protein
Escherichia coli
4.2.99.20
0.295
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R90A mutant protein
Escherichia coli
4.2.99.20
1.5
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D210A mutant protein
Escherichia coli
4.2.99.20
2.4
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147A mutant protein
Escherichia coli
4.2.99.20
6.1
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R124A mutant protein
Escherichia coli
4.2.99.20
10
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
S86A mutant protein
Escherichia coli
4.2.99.20
15
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
R168A mutant protein
Escherichia coli
4.2.99.20
180
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
Y85F mutant protein
Escherichia coli
4.2.99.20
220
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
W147F mutant protein
Escherichia coli
4.2.99.20
1400
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
D128A mutant protein
Escherichia coli
4.2.99.20
2200
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
K212A mutant protein
Escherichia coli
4.2.99.20
14000
-
(1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate
wild-type protein
Escherichia coli