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Literature summary extracted from

  • Qin, M.; Song, H.; Dai, X.; Chen, Y.; Guo, Z.
    Two active site arginines are critical determinants of substrate binding and catalysis in MenD a thiamine-dependent enzyme in menaquinone biosynthesis (2018), Biochem. J., 475, 3651-3667 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.2.1.9 expressed in Escherichia coli BL21(DE3) cells Escherichia coli

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.2.1.9 mutant enzyme R395K bound to thiamine diphosphate, hanging drop vapor diffusion method, using 0.2 M ammonium acetate, 0.06 M magnesium formate, 3% (w/v) polyethylene glycol 3350, and 12% (w/v) polyethylene glycol 10000 in 0.1 M Tris-HCl (pH 7.5). Mutant enzyme R395A bound to thiamine diphosphate, hanging drop vapor diffusion method, using 30% (v/v) Jeffamine M-600 (pH 7.0), 11.2% (w/v) polyethylene glycol 3350 and 0.16 M magnesium formate in 0.1 M HEPES (pH 7.5). Mutant enzyme R413A bound to thiamine diphosphate, hanging drop vapor diffusion method, using 0.16 M magnesium formate, 1% (w/v) tacsimate (pH 7.0), 14% (w/v) polyethylene glycol 3350, and 2% (w/v) polyethylene glycol MME 5000 in 0.02 M HEPES (pH 7.0) Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
2.2.1.9 Q118A the mutant is completely inactive Escherichia coli
2.2.1.9 R395A the mutant shows a 160 and 1000fold decrease in kcat/Km value with respect to 2-oxoglutarate and isochorismate, respectively, compared to the wild type enzyme Escherichia coli
2.2.1.9 R395K the mutant shows a 45 and 66fold decrease in kcat/Km value with respect to 2-oxoglutarate and isochorismate, respectively, compared to the wild type enzyme Escherichia coli
2.2.1.9 R395K/R413K the mutant shows a 6300 and 1300fold decrease in kcat/Km value with respect to 2-oxoglutarate and isochorismate, respectively, compared to the wild type enzyme Escherichia coli
2.2.1.9 R413A the mutant is completely inactive Escherichia coli
2.2.1.9 R413K the mutant shows a 270 and 37fold decrease in kcat/Km value with respect to 2-oxoglutarate and isochorismate, respectively, compared to the wild type enzyme Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.2.1.9 0.00008
-
 isochorismate wild type enzyme, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.00035
-
 isochorismate mutant enzyme R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.00082
-
 isochorismate mutant enzyme R395K/R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.0022
-
 isochorismate mutant enzyme R395K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.0029
-
 2-oxoglutarate wild type enzyme, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.016
-
 isochorismate mutant enzyme R395A, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.029
-
 2-oxoglutarate mutant enzyme R395A, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.067
-
 2-oxoglutarate mutant enzyme R395K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.115
-
 2-oxoglutarate mutant enzyme R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.172
-
 2-oxoglutarate mutant enzyme R395K/R413K, at pH 7.0 and 21°C Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.2.1.9 Mg2+ 5 mM used in assay conditions Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.2.1.9 isochorismate + 2-oxoglutarate Escherichia coli
-
 5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.2.1.9 Escherichia coli P17109
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.2.1.9 DEAE column chromatography and Sephacryl S-200 gel filtration Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.2.1.9 isochorismate + 2-oxoglutarate
-
 Escherichia coli 5-enolpyruvoyl-6-hydroxy-2-succinyl-cyclohex-3-ene-1-carboxylate + CO2
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.2.1.9 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase
-
 Escherichia coli
2.2.1.9 MenD
-
 Escherichia coli
2.2.1.9 SEPHCHC synthase
-
 Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.2.1.9 0.0025
-
 isochorismate mutant enzyme R395K/R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.0027
-
 2-oxoglutarate mutant enzyme R395K/R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.018
-
 2-oxoglutarate mutant enzyme R395A, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.038
-
 isochorismate mutant enzyme R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.043
-
 2-oxoglutarate mutant enzyme R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.065
-
 isochorismate mutant enzyme R395A, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.135
-
 isochorismate mutant enzyme R395K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.143
-
 2-oxoglutarate mutant enzyme R395K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.287
-
 2-oxoglutarate wild type enzyme, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.337
-
 isochorismate wild type enzyme, at pH 7.0 and 21°C Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
2.2.1.9 thiamine diphosphate
-
 Escherichia coli

General Information

EC Number General Information Comment Organism
2.2.1.9 metabolism the enzyme catalyzes an essential Stetter reaction in menaquinone (vitamin K2) biosynthesis via thiamine diphosphate (ThDP)-bound tetrahedral postdecarboxylation intermediates Escherichia coli

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.2.1.9 0.016
-
 2-oxoglutarate mutant enzyme R395K/R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.3
-
 isochorismate mutant enzyme R395K/R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.37
-
 2-oxoglutarate mutant enzyme R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 0.62
-
 2-oxoglutarate mutant enzyme R395A, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 2.2
-
 2-oxoglutarate mutant enzyme R395K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 4
-
 isochorismate mutant enzyme R395A, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 6.2
-
 isochorismate mutant enzyme R395K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 98.3
-
 2-oxoglutarate wild type enzyme, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 110
-
 isochorismate mutant enzyme R413K, at pH 7.0 and 21°C Escherichia coli
2.2.1.9 400
-
 isochorismate wild type enzyme, at pH 7.0 and 21°C Escherichia coli