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Literature summary for 2.2.1.9 extracted from

  • Dawson, A.; Chen, M.; Fyfe, P.K.; Guo, Z.; Hunter, W.N.
    Structure and reactivity of Bacillus subtilis MenD catalyzing the first committed step in menaquinone biosynthesis (2010), J. Mol. Biol., 401, 253-264.
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

Crystallization (Comment) Organism
to 2.35 A resolution, in complex with thiamine diphosphate and Mn2+. basic residues Arg32, Arg106, Arg409, Arg428, and Lys299 interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of the non-polar residues Ile489, Phe490, and Leu493 on one side of the active site. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of 2-oxoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate Bacillus subtilis

Protein Variants

Protein Variants Comment Organism
F490A 0.6% of wild-type activity Bacillus subtilis
I489A 1% of wild-type activity Bacillus subtilis
K299A 150% of wild-type activity Bacillus subtilis
L493A 80% of wild-type activity Bacillus subtilis
R106A mutation in the second subunit forming the active site, 10% of wild-type activity Bacillus subtilis
R32A mutation in the second subunit forming the active site, 30% of wild-type activity Bacillus subtilis
R409A 3% of wild-type activity Bacillus subtilis
R428A 1% of wild-type activity Bacillus subtilis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0077
-
2-oxoglutarate mutant R106A, pH 7.8, 23°C Bacillus subtilis
0.018
-
2-oxoglutarate mutant L493A, pH 7.8, 23°C Bacillus subtilis
0.022
-
2-oxoglutarate mutant K299A, pH 7.8, 23°C Bacillus subtilis
0.022
-
2-oxoglutarate mutant R32A, pH 7.8, 23°C Bacillus subtilis
0.022
-
2-oxoglutarate wild-type, pH 7.8, 23°C Bacillus subtilis
0.159
-
2-oxoglutarate mutant R409A, pH 7.8, 23°C Bacillus subtilis
0.274
-
2-oxoglutarate mutant F490A, pH 7.8, 23°C Bacillus subtilis
0.346
-
2-oxoglutarate mutant I489A, pH 7.8, 23°C Bacillus subtilis
0.514
-
2-oxoglutarate mutant R428A, pH 7.8, 23°C Bacillus subtilis

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
290000
-
gel filtration Bacillus subtilis

Organism

Organism UniProt Comment Textmining
Bacillus subtilis P23970
-
-

Reaction

Reaction Comment Organism Reaction ID
isochorismate + 2-oxoglutarate = 5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2 two-stage mechanism that is primarily driven by the chemical properties of the cofactor thiamine diphosphate Bacillus subtilis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
isochorismate + 2-oxoglutarate
-
Bacillus subtilis 5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2
-
?

Subunits

Subunits Comment Organism
tetramer dimer of dimer, crystallization data, 4 x 64100, calculated Bacillus subtilis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.01
-
2-oxoglutarate mutant R106A, pH 7.8, 23°C Bacillus subtilis
0.02
-
2-oxoglutarate mutant F490A, pH 7.8, 23°C Bacillus subtilis
0.05
-
2-oxoglutarate mutant R409A, pH 7.8, 23°C Bacillus subtilis
0.06
-
2-oxoglutarate mutant R428A, pH 7.8, 23°C Bacillus subtilis
0.08
-
2-oxoglutarate mutant R32A, pH 7.8, 23°C Bacillus subtilis
0.1
-
2-oxoglutarate mutant I489A, pH 7.8, 23°C Bacillus subtilis
0.17
-
2-oxoglutarate mutant L493A, pH 7.8, 23°C Bacillus subtilis
0.23
-
2-oxoglutarate wild-type, pH 7.8, 23°C Bacillus subtilis
0.37
-
2-oxoglutarate mutant K299A, pH 7.8, 23°C Bacillus subtilis

Cofactor

Cofactor Comment Organism Structure
thiamine diphosphate in absence of any residue that can act as a general acid/base the cofactor N4' atom is a critical component of the mechanism Bacillus subtilis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.07
-
2-oxoglutarate mutant F490A, pH 7.8, 23°C Bacillus subtilis
0.12
-
2-oxoglutarate mutant R428A, pH 7.8, 23°C Bacillus subtilis
0.27
-
2-oxoglutarate mutant I489A, pH 7.8, 23°C Bacillus subtilis
1.5
-
2-oxoglutarate mutant R106A, pH 7.8, 23°C Bacillus subtilis
2.7
-
2-oxoglutarate mutant R409A, pH 7.8, 23°C Bacillus subtilis
3.7
-
2-oxoglutarate mutant R32A, pH 7.8, 23°C Bacillus subtilis
8.3
-
2-oxoglutarate mutant L493A, pH 7.8, 23°C Bacillus subtilis
10.7
-
2-oxoglutarate wild-type, pH 7.8, 23°C Bacillus subtilis
16.7
-
2-oxoglutarate mutant K299A, pH 7.8, 23°C Bacillus subtilis