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Literature summary extracted from

  • Chen, Y.; Sun, Y.; Song, H.; Guo, Z.
    Structural basis for the ATP-dependent configuration of adenylation active site in Bacillus subtilis o-succinylbenzoyl-CoA synthetase (2015), J. Biol. Chem., 290, 23971-23983 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
6.2.1.26 drug development o-succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of antibiotics in the menaquinone biosynthesis Bacillus subtilis

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.2.1.26 recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Bacillus subtilis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
6.2.1.26 purified enzyme BsMenE, hanging drop vapor diffusion method, 1:1 mixture of a solution of protein at 10 mg/ml and a reservoir solution at pH 6.15 containing 8.1% w/v PEG 8000, 0.09 M cacodylate/HCl, 0.144 M calcium acetate, 12.6% glycerol, 0.049 M sodium phosphate monobasic, and 0.091 M dibasic potassium phosphate, a BsMenE-ATP-Mg2-complex is grown within a week in a 1:1 mixture of a solution of 10 mg/ml BsMenE protein preincubated with 3 mM ATP and 5 mM MgCl2 and a reservoir solution at pH 7.7 containing 7.2% v/v ethylene glycol, 9.9% w/v PEG 8000, 0.09 M HEPES, 3.5% v/v 2-methyl-2,4-pentanediol, and 0.01 M acetate, and a BsMenE-AMP complex grown from an 1:1 mixture of a solution of 10 mg/ml BsMenE protein preincubated with 3 mM AMP and mixed with a reservoir solution at pH 7.5 containing 0.10 M HEPES, 10% w/v PEG 6000, 5% v/v 2-methyl-2,4-pentadiol, 16°C, X-ray diffraction structure determination and analysis at 1.98-2.82 A resolution Bacillus subtilis

Protein Variants

EC Number Protein Variants Comment Organism
6.2.1.26 G154P site-directed mutagenesis, the mutant shows the same stability and circular dichroism spectra as the wild-type enzyme, but reduced catalytic activity Bacillus subtilis
6.2.1.26 G157P site-directed mutagenesis, the mutant shows the same stability and circular dichroism spectra as the wild-type enzyme, but reduced catalytic activity Bacillus subtilis
6.2.1.26 additional information mutation of the P-loop residues hydrogen-bonded to ATP reveals a crucial catalytic role of ATP-enzyme interaction Bacillus subtilis
6.2.1.26 R382A site-directed mutagenesis, the mutant shows the same stability and circular dichroism spectra as the wild-type enzyme, but reduced catalytic activity Bacillus subtilis
6.2.1.26 R382K site-directed mutagenesis, the mutant shows the same stability and circular dichroism spectra as the wild-type enzyme, but reduced catalytic activity Bacillus subtilis
6.2.1.26 T152A site-directed mutagenesis, the mutant shows the same stability and circular dichroism spectra as the wild-type enzyme, but reduced catalytic activity Bacillus subtilis
6.2.1.26 T155A site-directed mutagenesis, the mutant shows the same stability and circular dichroism spectra as the wild-type enzyme, but reduced catalytic activity Bacillus subtilis
6.2.1.26 T155A/T156A site-directed mutagenesis, the mutant shows the same stability and circular dichroism spectra as the wild-type enzyme, but reduced catalytic activity Bacillus subtilis
6.2.1.26 T156A site-directed mutagenesis, the mutant shows the same stability and circular dichroism spectra as the wild-type enzyme, but reduced catalytic activity Bacillus subtilis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.2.1.26 additional information
-
 additional information single-substrate kinetics Bacillus subtilis
6.2.1.26 0.024
-
 ATP pH 7.5, 24°C, recombinant wild-type enzyme Bacillus subtilis
6.2.1.26 0.044
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant wild-type enzyme Bacillus subtilis
6.2.1.26 0.057
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant R382A Bacillus subtilis
6.2.1.26 0.066
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant R382K Bacillus subtilis
6.2.1.26 0.08
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant G157P Bacillus subtilis
6.2.1.26 0.087
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant T155A Bacillus subtilis
6.2.1.26 0.12
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant G154P Bacillus subtilis
6.2.1.26 0.14
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant T156A Bacillus subtilis
6.2.1.26 0.17
-
 CoA pH 7.5, 24°C, recombinant mutant T155A/T156A Bacillus subtilis
6.2.1.26 0.21
-
 CoA pH 7.5, 24°C, recombinant mutant T156A Bacillus subtilis
6.2.1.26 0.24
-
 CoA pH 7.5, 24°C, recombinant wild-type enzyme Bacillus subtilis
6.2.1.26 0.25
-
 CoA pH 7.5, 24°C, recombinant mutant G157P Bacillus subtilis
6.2.1.26 0.26
-
 CoA pH 7.5, 24°C, recombinant mutant T152A Bacillus subtilis
6.2.1.26 0.27
-
 CoA pH 7.5, 24°C, recombinant mutant G154P Bacillus subtilis
6.2.1.26 0.28
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant T155A/T156A Bacillus subtilis
6.2.1.26 0.41
-
 CoA pH 7.5, 24°C, recombinant mutant T155A Bacillus subtilis
6.2.1.26 0.88
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant T152A Bacillus subtilis
6.2.1.26 2.3
-
 ATP pH 7.5, 24°C, recombinant mutant T156A Bacillus subtilis
6.2.1.26 2.7
-
 CoA pH 7.5, 24°C, recombinant mutant R382A Bacillus subtilis
6.2.1.26 3
-
 CoA pH 7.5, 24°C, recombinant mutant R382K Bacillus subtilis
6.2.1.26 3.17
-
 ATP pH 7.5, 24°C, recombinant mutant G157P Bacillus subtilis
6.2.1.26 3.3
-
 ATP pH 7.5, 24°C, recombinant mutant T155A/T156A Bacillus subtilis
6.2.1.26 3.4
-
 ATP pH 7.5, 24°C, recombinant mutant G154P Bacillus subtilis
6.2.1.26 3.5
-
 ATP pH 7.5, 24°C, recombinant mutant T152A Bacillus subtilis
6.2.1.26 4.2
-
 ATP pH 7.5, 24°C, recombinant mutant T155A Bacillus subtilis
6.2.1.26 4.67
-
 ATP pH 7.5, 24°C, recombinant mutant R382K Bacillus subtilis
6.2.1.26 8.5
-
 ATP pH 7.5, 24°C, recombinant mutant R382A Bacillus subtilis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.2.1.26 Mg2+ required Bacillus subtilis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.2.1.26 ATP + 2-succinylbenzoate + CoA Bacillus subtilis
-
 AMP + diphosphate + 4-(2-carboxyphenyl)-4-oxobutanoyl-CoA
-
 ?
6.2.1.26 ATP + 2-succinylbenzoate + CoA Bacillus subtilis 168
-
 AMP + diphosphate + 4-(2-carboxyphenyl)-4-oxobutanoyl-CoA
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.2.1.26 Bacillus subtilis P23971
-
-
6.2.1.26 Bacillus subtilis 168 P23971
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
6.2.1.26 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Bacillus subtilis

Reaction

EC Number Reaction Comment Organism Reaction ID
6.2.1.26 ATP + 2-succinylbenzoate + CoA = AMP + diphosphate + 4-(2-carboxyphenyl)-4-oxobutanoyl-CoA the enzyme adopts an ordered bi uni uni bi ping-pong mechanism, which involves ATP as the first binding substrate and a large conformational change during the catalysis. In MenE catalysis, tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site create a new binding site for the carboxylate substrate. Analysis of the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylateforming enzymes, open-closed conformational change in ATP configuration of the adenylation active site Bacillus subtilis
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.2.1.26 ATP + 2-succinylbenzoate + CoA
-
 Bacillus subtilis AMP + diphosphate + 4-(2-carboxyphenyl)-4-oxobutanoyl-CoA
-
 ?
6.2.1.26 ATP + 2-succinylbenzoate + CoA
-
 Bacillus subtilis 168 AMP + diphosphate + 4-(2-carboxyphenyl)-4-oxobutanoyl-CoA
-
 ?

Subunits

EC Number Subunits Comment Organism
6.2.1.26 homodimer
-
 Bacillus subtilis
6.2.1.26 More structure motif sequence comparisons Bacillus subtilis

Synonyms

EC Number Synonyms Comment Organism
6.2.1.26 BsMenE
-
 Bacillus subtilis
6.2.1.26 MenE
-
 Bacillus subtilis
6.2.1.26 o-succinylbenzoyl-CoA synthetase
-
 Bacillus subtilis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.2.1.26 24
-
 assay at Bacillus subtilis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
6.2.1.26 0.045
-
 ATP pH 7.5, 24°C, recombinant mutant T155A/T156A Bacillus subtilis
6.2.1.26 0.045
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant T155A/T156A Bacillus subtilis
6.2.1.26 0.05
-
 CoA pH 7.5, 24°C, recombinant mutant T155A/T156A Bacillus subtilis
6.2.1.26 0.077
-
 ATP pH 7.5, 24°C, recombinant mutant T155A Bacillus subtilis
6.2.1.26 0.103
-
 CoA pH 7.5, 24°C, recombinant mutant T155A Bacillus subtilis
6.2.1.26 0.125
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant T155A Bacillus subtilis
6.2.1.26 0.135
-
 ATP pH 7.5, 24°C, recombinant mutant T152A Bacillus subtilis
6.2.1.26 0.137
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant T152A Bacillus subtilis
6.2.1.26 0.145
-
 CoA pH 7.5, 24°C, recombinant mutant T156A Bacillus subtilis
6.2.1.26 0.15
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant R382A Bacillus subtilis
6.2.1.26 0.162
-
 CoA pH 7.5, 24°C, recombinant mutant R382K Bacillus subtilis
6.2.1.26 0.163
-
 ATP pH 7.5, 24°C, recombinant mutant R382K Bacillus subtilis
6.2.1.26 0.206
-
 CoA pH 7.5, 24°C, recombinant mutant T152A Bacillus subtilis
6.2.1.26 0.213
-
 ATP pH 7.5, 24°C, recombinant mutant T156A Bacillus subtilis
6.2.1.26 0.213
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant T156A Bacillus subtilis
6.2.1.26 0.218
-
 ATP pH 7.5, 24°C, recombinant mutant R382A Bacillus subtilis
6.2.1.26 0.228
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant R382K Bacillus subtilis
6.2.1.26 0.283
-
 CoA pH 7.5, 24°C, recombinant mutant R382A Bacillus subtilis
6.2.1.26 1.65
-
 ATP pH 7.5, 24°C, recombinant mutant G154P Bacillus subtilis
6.2.1.26 2.17
-
 CoA pH 7.5, 24°C, recombinant mutant G154P Bacillus subtilis
6.2.1.26 2.17
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant G154P Bacillus subtilis
6.2.1.26 3
-
 ATP pH 7.5, 24°C, recombinant mutant G157P Bacillus subtilis
6.2.1.26 4.3
-
 CoA pH 7.5, 24°C, recombinant mutant G157P Bacillus subtilis
6.2.1.26 46.67
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant mutant G157P Bacillus subtilis
6.2.1.26 105
-
 ATP pH 7.5, 24°C, recombinant wild-type enzyme Bacillus subtilis
6.2.1.26 120
-
 2-succinylbenzoate pH 7.5, 24°C, recombinant wild-type enzyme Bacillus subtilis
6.2.1.26 121.7
-
 CoA pH 7.5, 24°C, recombinant wild-type enzyme Bacillus subtilis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.2.1.26 7.5
-
 assay at Bacillus subtilis

Cofactor

EC Number Cofactor Comment Organism Structure
6.2.1.26 ATP binding structure and ATP-enzyme interaction analysis Bacillus subtilis

General Information

EC Number General Information Comment Organism
6.2.1.26 metabolism o-succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme in the menaquinone biosynthesis Bacillus subtilis
6.2.1.26 additional information in MenE catalysis, tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site create a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and in-line alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling, overview. The ATP-enzyme interaction is suggested to play a crucial catalytic role. positioning and catalytic role of a conserved lysine residue in stabilization of the transition state, molecular docking, overview Bacillus subtilis
6.2.1.26 physiological function ATP-dependent configuration of adenylation active site in o-succinylbenzoyl-CoA synthetase Bacillus subtilis