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Literature summary for 5.4.99.14 extracted from

  • Marin, M.; Heinz, D.W.; Pieper, D.H.; Klink, B.U.
    Crystal structure and catalytic mechanism of 4-methylmuconolactone methylisomerase (2009), J. Biol. Chem., 284, 32709-32716.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli JM109 cells Pseudomonas reinekei

Crystallization (Commentary)

Crystallization (Comment) Organism
in complex with (4S)-4-methylmuconolactone and (4S)-3-methylmuconolactone, sitting drop vapor diffusion method, at 19°C, using 25% (w/v) polyethylene glycol 1500, 0.1 M MMT buffer, pH 5 (DL-malic acid, MES, and Tris base in a 1:2:2 molar ratio) Pseudomonas reinekei

Protein Variants

Protein Variants Comment Organism
C67S incubation of variant C67S with 4-chloromercuribenzoate does not significantly decrease its activity (above 65% of activity remaining after a 30-min incubation) Pseudomonas reinekei
H26A the mutant shows a marked decrease in catalytic activity (kcat) by two orders of magnitude Pseudomonas reinekei
H52A the mutation results in an increase in Km by a factor of 9, but only a small change in kcat Pseudomonas reinekei
Y39F the mutant displays reduced activity compared to the wild type enzyme Pseudomonas reinekei

Inhibitors

Inhibitors Comment Organism Structure
4-chloromercuribenzoate remaining activity of 6% after incubation with 0.02 mM 4-chloromercuribenzoate for 30 min Pseudomonas reinekei

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.045
-
(4S)-4-methylmuconolactone mutant enzyme Y39F, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei
0.052
-
(4S)-4-methylmuconolactone wild type enzyme, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei
0.0972
-
(4S)-4-methylmuconolactone mutant enzyme C67S, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei
0.4583
-
(4S)-4-methylmuconolactone mutant enzyme H52A, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei

Organism

Organism UniProt Comment Textmining
Pseudomonas reinekei C5MR76
-
-

Purification (Commentary)

Purification (Comment) Organism
Strep-Tactin-Sepharose column chromatography Pseudomonas reinekei

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
28.8
-
toward 1-methylbis-lactone, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei
57.3
-
toward (4S)-4-methylmuconolactone, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(4S)-4-methylmuconolactone
-
Pseudomonas reinekei (4S)-3-methylmuconolactone
-
?
1-methylbis-lactone
-
Pseudomonas reinekei ?
-
?
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
-
Pseudomonas reinekei 4-Carboxymethyl-3-methylbut-2-en-1,4-olide
-
?
additional information activity with 3-methyl-cis,cis-muconate is below the detection limit Pseudomonas reinekei ?
-
?

Subunits

Subunits Comment Organism
homodimer x-ray crystallography Pseudomonas reinekei

Synonyms

Synonyms Comment Organism
4-Methylmuconolactone methylisomerase
-
Pseudomonas reinekei
MLMI
-
Pseudomonas reinekei

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.8
-
(4S)-4-methylmuconolactone mutant enzyme Y39F, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei
7.3
-
(4S)-4-methylmuconolactone mutant enzyme C67S, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei
24.2
-
(4S)-4-methylmuconolactone wild type enzyme, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei
47.2
-
(4S)-4-methylmuconolactone mutant enzyme H52A, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.3 6.5
-
Pseudomonas reinekei

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
100
-
(4S)-4-methylmuconolactone mutant enzyme C67S, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei
100
-
(4S)-4-methylmuconolactone mutant enzyme H52A, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei
100
-
(4S)-4-methylmuconolactone mutant enzyme Y39F, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei
500
-
(4S)-4-methylmuconolactone wild type enzyme, in phosphate buffer, 50 mM, pH 7.3 Pseudomonas reinekei