Information on EC 5.4.99.14 - 4-Carboxymethyl-4-methylbutenolide mutase

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The expected taxonomic range for this enzyme is: Bacteria

EC NUMBER
COMMENTARY
5.4.99.14
-
RECOMMENDED NAME
GeneOntology No.
4-Carboxymethyl-4-methylbutenolide mutase
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
4-Carboxymethyl-4-methylbut-2-en-1,4-olide = 4-carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
-
-
-
-
4-Carboxymethyl-4-methylbut-2-en-1,4-olide = 4-carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
isomerization does not involve actual methyl migration, but proceeds via the 1-methylbislactone
-
4-Carboxymethyl-4-methylbut-2-en-1,4-olide = 4-carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
mechanism
-
4-Carboxymethyl-4-methylbut-2-en-1,4-olide = 4-carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
isomerization does not involve actual methyl migration, but proceeds via the 1-methylbislactone
Rhodococcus rhodochrous N75
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
intramolecular transfer reaction
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
4-methylcatechol degradation (ortho cleavage)
-
-
Toluene degradation
-
-
Microbial metabolism in diverse environments
-
-
SYSTEMATIC NAME
IUBMB Comments
4-Carboxymethyl-4-methylbut-2-en-1,4-olide methylmutase
-
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4-Methyl-3-enelactone methyl isomerase
-
-
-
-
4-Methylmuconolactone methylisomerase
-
-
-
-
4-Methylmuconolactone methylisomerase
-
4-Methylmuconolactone methylisomerase
Cupriavidus necator JMP134
-
-
4-Methylmuconolactone methylisomerase
-
-
4-Methylmuconolactone methylisomerase
-
4-Methylmuconolactone methylisomerase
Pseudomonas reinekei MT1
-
-
Isomerase, 4-methyl-2-enelactone
-
-
-
-
methymuconolactone isomerase
-
-
methymuconolactone isomerase
Pseudomonas reinekei MT1
-
-
-
MLMI
Cupriavidus necator JMP134
-
-
MLMI
Pseudomonas reinekei MT1
-
-
MmlJ
Pseudomonas reinekei MT1
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
115300-03-3
-
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
basonym Alcaligenes eutrophus; JMP 134, cloned into Pseudomonas sp. B13 FR1
-
-
Manually annotated by BRENDA team
gene mmlI
UniProt
Manually annotated by BRENDA team
Cupriavidus necator JMP134
gene mmlI
UniProt
Manually annotated by BRENDA team
Gordonia rubripertincta N657
N657
-
-
Manually annotated by BRENDA team
strain MT1
-
-
Manually annotated by BRENDA team
strain MT1
UniProt
Manually annotated by BRENDA team
Pseudomonas reinekei MT1
strain MT1
-
-
Manually annotated by BRENDA team
Pseudomonas reinekei MT1
strain MT1
UniProt
Manually annotated by BRENDA team
Rhodococcus rhodochrous N5
N5
-
-
Manually annotated by BRENDA team
Rhodococcus rhodochrous N75
N75
-
-
Manually annotated by BRENDA team
Rhodococcus ruber N75
N75
-
-
Manually annotated by BRENDA team
strains BCN1, BCN2 and 4PH1
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
physiological function
the enzyme is involved in the modified ortho-cleavage pathway and methylmuconolactone degradation of Ralstonia eutropha strain JMP134
physiological function
Cupriavidus necator JMP134
-
the enzyme is involved in the modified ortho-cleavage pathway and methylmuconolactone degradation of Ralstonia eutropha strain JMP134
-
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(4R,5R)-5-chloro-3-methylmuconolactone
trans-3-methyldienelactone
show the reaction diagram
Cupriavidus necator, Cupriavidus necator JMP134
-
-
r
(4S)-4-methylmuconolactone
(4S)-3-methylmuconolactone
show the reaction diagram
Pseudomonas reinekei, Pseudomonas reinekei MT1
-
-
?
(4S,5S)-5-chloro-3-methylmuconolactone
trans-3-methyldienelactone
show the reaction diagram
Cupriavidus necator, Cupriavidus necator JMP134
-
-
r
1-methylbis-lactone
?
show the reaction diagram
Pseudomonas reinekei, Pseudomonas reinekei MT1
-
-
-
?
1-Methylbislactone
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
-
-
-
-
1-Methylbislactone
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
-
i.e. 1-methyl-3,7-dioxo-2,6-dioxabicyclo[3.3.0]octane
-
-
1-Methylbislactone
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
Rhodococcus rhodochrous N75
-
-
-
-
3-methylmuconolactone
4-methyl-3-oxoadipate enollactone
show the reaction diagram
Pseudomonas reinekei, Pseudomonas reinekei MT1
-
-
-
?
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
-
-
-
-
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
-
-
-
-
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
-
-
-
-
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
-
-
-
?
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
-
-
?
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
-
highly specific, accepts only (+)-(4S)-4-methylmuconolactone, i.e. 4-carboxymethyl-4-methylbut-2-en-1,4-olide and the putative reaction intermediate 1-methylbislactone
i.e. (-)-(4S)-3-methylmuconolactone
-
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
Pseudomonas reinekei MT1
-
-
-
?
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
Pseudomonas reinekei MT1
-
-
?
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
Rhodococcus rhodochrous N5, Gordonia rubripertincta N657
-
-
-
-
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
Rhodococcus rhodochrous N75
-
highly specific, accepts only (+)-(4S)-4-methylmuconolactone, i.e. 4-carboxymethyl-4-methylbut-2-en-1,4-olide and the putative reaction intermediate 1-methylbislactone
i.e. (-)-(4S)-3-methylmuconolactone
-
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
4-Carboxymethyl-3-methylbut-2-en-1,4-olide
show the reaction diagram
Rhodococcus ruber N75
-
-
-
-
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
?
show the reaction diagram
-
enzyme of the modified 3-oxoadipate pathway in the Gram-negative bacterium Alcaligenes eutrophus JMP 134
-
-
-
4-methyl-muconolactone
3-methyl-muconolactone
show the reaction diagram
Cupriavidus necator, Cupriavidus necator JMP134
-
-
r
additional information
?
-
-
not: isomeric methyl-substituted 4-carboxymethylbut-2-en-4-olides
-
-
-
additional information
?
-
Pseudomonas reinekei, Pseudomonas reinekei MT1
activity with 3-methyl-cis,cis-muconate is below the detection limit
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
4-Carboxymethyl-4-methylbut-2-en-1,4-olide
?
show the reaction diagram
-
enzyme of the modified 3-oxoadipate pathway in the Gram-negative bacterium Alcaligenes eutrophus JMP 134
-
-
-
4-methyl-muconolactone
3-methyl-muconolactone
show the reaction diagram
Cupriavidus necator, Cupriavidus necator JMP134
O51799
-
-
r
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(+)-muconolactone
-
-
(-)1-Methyl-8-bromobislactone
-
weak
2,4-Dimethyl-2-enelactone
-
weak
-
2-Bromo-4-methylbutan-1,4-olide
-
weak
2-Bromobutan-1,4-olide
-
-
2-Methyl-2-enelactone
-
weak
-
2-Methylbutan-1,4-olide
-
-
3-Methyl-2-enelactone
-
weak
-
4-carboxymethylbut-2-en-4-olide
-
-
4-chloromercuribenzoate
remaining activity of 6% after incubation with 0.02 mM 4-chloromercuribenzoate for 30 min
4-chloromercuribenzoate
inhibition is reversible by DTT
4-methyl-2-enelactone
-
weak
-
4-Methylbut-3-en-1,4-olide
-
angelicalactone
4-methylbutan-1,4-olide
-
-
trans-dienelactone
-
weak
additional information
-
EDTA
-
additional information
-
not: isomeric methyl-substituted 4-carboxymethylbut-2-en-4-olides
-
additional information
no inhibition by EDTA
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.045
(4S)-4-methylmuconolactone
mutant enzyme Y39F, in phosphate buffer, 50 mM, pH 7.3
0.052
(4S)-4-methylmuconolactone
wild type enzyme, in phosphate buffer, 50 mM, pH 7.3
0.0972
(4S)-4-methylmuconolactone
mutant enzyme C67S, in phosphate buffer, 50 mM, pH 7.3
0.4583
(4S)-4-methylmuconolactone
mutant enzyme H52A, in phosphate buffer, 50 mM, pH 7.3
0.028
1-methyl-3,7-dioxo-2,6-dioxabicyclo[3.3.0]octane
-
-
0.17
1-methyl-bislactone
-
-
0.176
4-carboxymethyl-4-methylbut-2-en-1,4-olide
-
-
0.23
4-carboxymethyl-4-methylbut-2-en-1,4-olide
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
5.8
(4S)-4-methylmuconolactone
Pseudomonas reinekei
C5MR76
mutant enzyme Y39F, in phosphate buffer, 50 mM, pH 7.3
7.3
(4S)-4-methylmuconolactone
Pseudomonas reinekei
C5MR76
mutant enzyme C67S, in phosphate buffer, 50 mM, pH 7.3
24.2
(4S)-4-methylmuconolactone
Pseudomonas reinekei
C5MR76
wild type enzyme, in phosphate buffer, 50 mM, pH 7.3
47.2
(4S)-4-methylmuconolactone
Pseudomonas reinekei
C5MR76
mutant enzyme H52A, in phosphate buffer, 50 mM, pH 7.3
3.75
1-methyl-3,7-dioxo-2,6-dioxabicyclo[3.3.0]octane
Cupriavidus necator
-
-
98.5
4-carboxymethyl-4-methylbut-2-en-1,4-olide
Cupriavidus necator
-
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
100
(4S)-4-methylmuconolactone
Pseudomonas reinekei
C5MR76
mutant enzyme C67S, in phosphate buffer, 50 mM, pH 7.3; mutant enzyme H52A, in phosphate buffer, 50 mM, pH 7.3; mutant enzyme Y39F, in phosphate buffer, 50 mM, pH 7.3
8851
500
(4S)-4-methylmuconolactone
Pseudomonas reinekei
C5MR76
wild type enzyme, in phosphate buffer, 50 mM, pH 7.3
8851
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
28.8
toward 1-methylbis-lactone, in phosphate buffer, 50 mM, pH 7.3
57.3
toward (4S)-4-methylmuconolactone, in phosphate buffer, 50 mM, pH 7.3
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6 - 8.5
-
active in this range, rapid decrease of activity above pH 8.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
Gordonia rubripertincta N657, Rhodococcus rhodochrous N5, Rhodococcus rhodochrous N75, Rhodococcus ruber N75
-
-
-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
Rhodococcus rhodochrous N75
-
-
-
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25000
gel filtration
717634
75500
-
Rhodococcus rhodochrous, gel filtration
3482
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homodimer
x-ray crystallography
homodimer
2 * 12900, about, sequence calculation
homodimer
Cupriavidus necator JMP134
-
2 * 12900, about, sequence calculation
-
homodimer
Pseudomonas reinekei MT1
-
x-ray crystallography
-
monomer
-
1 * 40000, Alcaligenes eutrophus (JMP 134, cloned into Pseudomonas sp. B13 FR1), SDS-PAGE
tetramer
-
4 * 17000, Rhodococcus rhodochrous, SDS-PAGE
tetramer
Rhodococcus rhodochrous N75
-
4 * 17000, Rhodococcus rhodochrous, SDS-PAGE
-
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with (4S)-4-methylmuconolactone and (4S)-3-methylmuconolactone, sitting drop vapor diffusion method, at 19C, 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)
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Chelating agents like EDTA or thiol-group modifying reagents like DTT stabilize
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C or -20C, highly purified enzyme is rather unstable
-
4C, 24 h, 10% loss of activity, highly purified enzyme, 5% loss of activity with addition of chelating agents like EDTA or thiol-group modifying reagents like DTT
-
-30C, slow loss of activity on prolonged storage
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
JMP 134, cloned into Pseudomonas sp. B13 FR1
-
MonoQ column chromatography
-
Strep-Tactin-Sepharose column chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene mmlI, DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain BL21(DE3)
expressed in Escherichia coli JM109 cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
C67S
incubation of variant C67S with 4-chloromercuribenzoate does not significantly decrease its activity (above 65% of activity remaining after a 30-min incubation)
H26A
the mutant shows a marked decrease in catalytic activity (kcat) by two orders of magnitude
H52A
the mutation results in an increase in Km by a factor of 9, but only a small change in kcat
Y39F
the mutant displays reduced activity compared to the wild type enzyme
C67S
Pseudomonas reinekei MT1
-
incubation of variant C67S with 4-chloromercuribenzoate does not significantly decrease its activity (above 65% of activity remaining after a 30-min incubation)
-
H26A
Pseudomonas reinekei MT1
-
the mutant shows a marked decrease in catalytic activity (kcat) by two orders of magnitude
-
H52A
Pseudomonas reinekei MT1
-
the mutation results in an increase in Km by a factor of 9, but only a small change in kcat
-
Y39F
Pseudomonas reinekei MT1
-
the mutant displays reduced activity compared to the wild type enzyme
-