Literature summary for 6.2.1.26 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 .

Application

Application	Comment	Organism
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)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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
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
additional information	mutation of the P-loop residues hydrogen-bonded to ATP reveals a crucial catalytic role of ATP-enzyme interaction	Bacillus subtilis
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
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
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
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
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
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]

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

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Bacillus subtilis

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Bacillus subtilis	P23971	-	-
Bacillus subtilis 168	P23971	-	-

Purification (Commentary)

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

Reaction

Reaction	Comment	Organism	Reaction ID
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)

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

Subunits

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

Synonyms

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

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

General Information

General Information	Comment	Organism
metabolism	o-succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme in the menaquinone biosynthesis	Bacillus subtilis
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
physiological function	ATP-dependent configuration of adenylation active site in o-succinylbenzoyl-CoA synthetase	Bacillus subtilis