Information on EC 3.3.2.8 - limonene-1,2-epoxide hydrolase

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The expected taxonomic range for this enzyme is: Rhodococcus erythropolis

EC NUMBER
COMMENTARY
3.3.2.8
-
RECOMMENDED NAME
GeneOntology No.
limonene-1,2-epoxide hydrolase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
1,2-epoxymenth-8-ene + H2O = menth-8-ene-1,2-diol
show the reaction diagram
catalyzes simultaneous sequential and enantioconvergent epoxide conversion
-
1,2-epoxymenth-8-ene + H2O = menth-8-ene-1,2-diol
show the reaction diagram
one-step mechanism
-
1,2-epoxymenth-8-ene + H2O = menth-8-ene-1,2-diol
show the reaction diagram
mechanism involves a concerted general acid catalysis step involving the Asp101-Arg99-Asp132 triad. The water molecule acting as nucleophilic reagent moves to the more substituted oxirane carbon atom, its hydrogen atom transfers to Asp132, and hydroxyl attacks at C1. Meanwhile, Asp101 donates a proton to the epoxide oxygen opening the oxirane ring. This process has an energy barrier of 16.9 kcal/mol and an endothermicity of 8.2 kcal/mol, and yields (1R,2R,4S)-limonene-1,2-diol as product. Activation barriers of 16.9 and 25.1 kcal/mol are calculated at the B3LYP/6-31G(d,p)//CHARMM level for nucleophilic attack on the more and less substituted epoxide carbons, respectively
-
1,2-epoxymenth-8-ene + H2O = menth-8-ene-1,2-diol
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
hydrolysis of epoxide
-
-
-
-
PATHWAY
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
Limonene and pinene degradation
-
limonene degradation I (D-limonene)
-
limonene degradation II (L-limonene)
-
SYSTEMATIC NAME
IUBMB Comments
1,2-epoxymeth-8-ene hydrolase
Involved in the monoterpene degradation pathway of the actinomycete Rhodococcus erythropolis. The enzyme hydrolyses several alicyclic and 1-methyl-substituted epoxides, such as 1-methylcyclohexene oxide, indene oxide and cyclohexene oxide. It differs from the previously described epoxide hydrolases [EC 3.3.2.4 (trans-epoxysuccinate hydrolase), EC 3.3.2.6 (leukotriene-A4 hydrolase), EC 3.3.2.7 (hepoxilin-epoxide hydrolase), EC 3.3.2.9 (microsomal epoxide hydrolase) and EC 3.3.2.10 (soluble epoxide hydrolase)] as it is not inhibited by 2-bromo-4'-nitroacetophenone, diethyl dicarbonate, 4-fluorochalcone oxide or 1,10-phenanthroline. Both enantiomers of menth-8-ene-1,2-diol [i.e. (1R,2R,4S)-menth-8-ene-1,2-diol and (1S,2S,4R)-menth-8-ene-1,2-diol] are metabolized.
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
limonene oxide hydrolase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
216503-88-7
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
enzyme is induced if cells are grown on monoterpenes
-
-
Manually annotated by BRENDA team
Rhodococcus erythropolis DCL 14
-
-
-
Manually annotated by BRENDA team
Rhodococcus erythropolis DCL14
DCL14
SwissProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(1R,2S)-1-methylcyclohexane oxide + H2O
(1S,2S)-1-methylcyclohexane-1,2-diol
show the reaction diagram
Rhodococcus erythropolis, Rhodococcus erythropolis DCL14
Q9ZAG3
-
-
-
ir
(1R,2S,4R)-limonene-1,2-epoxide + H2O
(1S,2S,4R)-limonene-1,2-diol
show the reaction diagram
-
-
optically pure, diastereomeric excess above 99%
?
(1R,2S,4R)-limonene-1,2-epoxide + H2O
(1S,2S,4R)-limonene-1,2-diol
show the reaction diagram
Rhodococcus erythropolis, Rhodococcus erythropolis DCL14
Q9ZAG3
the reaction mechanism involves epoxide protonation by Asp109, nucleophilic attack by water, and abstraction of a proton from water by Asp132. The isopropenyl group plays a crucial role because it restricts the half-chair conformation to one of the two possible helicities. In this conformation, attack on the different epoxide carbons will lead to either a chair-like or a twist-boat transition state structure, the latter resulting in a higher barrier. The regioselectivity is thus governed by conformational and not electronic factors
-
-
ir
(1R,2S,4S)-limonene-1,2-epoxide + H2O
(1R,2R,4S)-limonene-1,2-diol
show the reaction diagram
-
-
optically pure, diastereomeric excess above 99%
?
(1R,2S,4S)-limonene-1,2-epoxide + H2O
(1R,2R,4S)-limonene-1,2-diol
show the reaction diagram
Rhodococcus erythropolis, Rhodococcus erythropolis DCL14
Q9ZAG3
the reaction mechanism involves epoxide protonation by Asp109, nucleophilic attack by water, and abstraction of a proton from water by Asp132. The isopropenyl group plays a crucial role because it restricts the half-chair conformation to one of the two possible helicities. In this conformation, attack on the different epoxide carbons will lead to either a chair-like or a twist-boat transition state structure, the latter resulting in a higher barrier. The regioselectivity is thus governed by conformational and not electronic factors
-
-
ir
(1S,2R)-1-methylcyclohexane oxide + H2O
(1R,2R)-1-methylcyclohexane-1,2-diol
show the reaction diagram
Q9ZAG3
-
-
-
ir
(1S,2R,4R)-limonene-1,2-epoxide + H2O
(1S,2S,4R)-limonene-1,2-diol
show the reaction diagram
-
-
optically pure, diastereomeric excess above 99%
?
(1S,2R,4R)-limonene-1,2-epoxide + H2O
(1S,2S,4R)-limonene-1,2-diol
show the reaction diagram
Rhodococcus erythropolis, Rhodococcus erythropolis DCL14
Q9ZAG3
the reaction mechanism involves epoxide protonation by Asp109, nucleophilic attack by water, and abstraction of a proton from water by Asp132. The isopropenyl group plays a crucial role because it restricts the half-chair conformation to one of the two possible helicities. In this conformation, attack on the different epoxide carbons will lead to either a chair-like or a twist-boat transition state structure, the latter resulting in a higher barrier. The regioselectivity is thus governed by conformational and not electronic factors
-
-
ir
(1S,2R,4S)-limonene-1,2-epoxide + H2O
(1R,2R,4S)-limonene-1,2-diol
show the reaction diagram
Q9ZAG3
the reaction mechanism involves epoxide protonation by Asp109, nucleophilic attack by water, and abstraction of a proton from water by Asp132. The isopropenyl group plays a crucial role because it restricts the half-chair conformation to one of the two possible helicities. In this conformation, attack on the different epoxide carbons will lead to either a chair-like or a twist-boat transition state structure, the latter resulting in a higher barrier. The regioselectivity is thus governed by conformational and not electronic factors
-
-
ir
(1S2R4S)-limonene-1,2-epoxide + H2O
(1R,2R,4S)-limonene-1,2-diol
show the reaction diagram
-
-
optically pure, diastereomeric excess above 99%
?
1,2-epoxy-2,6-dimethyl-5-heptane + H2O
(2S)-2,6-dimethyl-5-heptene-1,2-diol
show the reaction diagram
-
-
-
?
1,2-epoxy-2-methyl-6-heptene + H2O
(2S)-2-methyl-6-heptene-1,2-diol
show the reaction diagram
-
-
-
?
1,2-epoxy-2-methylheptane + H2O
(2S)-2-methylheptane-1,2-diol
show the reaction diagram
-
-
-
?
1,2-epoxy-3-benzyl-2-methylpropane + H2O
(2S)-3-benzyl-2-methylpropane-1,2-diol
show the reaction diagram
-
-
-
?
1-methylcyclohexene oxide + H2O
(1S,2S)-1-methylcyclohexane-1,2-diol
show the reaction diagram
-
-
-
?
cyclohexene oxide + H2O
(1S,2S)-trans cyclohexanediol + (1R,2R)-trans-cyclohexanediol
show the reaction diagram
-
-
-
?
cyclopentene-oxide + H2O
(R,R)-cyclopentene-1,2-diol + (S,S)-cyclopentene-1,2-diol
show the reaction diagram
Rhodococcus erythropolis, Rhodococcus erythropolis DCL 14
-
-
wild-type, 72% conversion, (R,R)-product with 14% enantiomeric excess
-
?
indene oxide + H2O
indane-1,2-diol
show the reaction diagram
-
-
-
?
limonene-1,2-eopxide + H2O
limonene-1,2-diol
show the reaction diagram
Rhodococcus erythropolis, Rhodococcus erythropolis DCL14
Q9ZAG3
part of limonene degradation pathway which allows the organism to grow on limone as sole source of carbon and energy
-
-
ir
limonene-1,2-epoxide + H2O
limonene-1,2-diol
show the reaction diagram
Q9ZAG3
-
-
-
ir
rac-1-methyl-7-oxabicyclo[4.1.0]heptane + H2O
(1S,2S)-1-methylcyclohexane-1,2-diol + (1R,6S)-1-methyl-7-oxabicyclo[4.1.0]heptane
show the reaction diagram
Rhodococcus erythropolis, Rhodococcus erythropolis DCL 14
-
-
wild-type, 99% conversion, 19% enantiomeric excess for (1S,2S)-product
-
-
rac-2-(phenoxymethyl)oxirane + H2O
(2S)-2-(phenoxymethyl)oxirane + (2R)-3-phenoxypropane-1,2-diol
show the reaction diagram
Rhodococcus erythropolis, Rhodococcus erythropolis DCL 14
-
-
wild-type, 33% conversion, 37% enantiomeric excess
-
?
styrene-7,8-oxide + H2O
styrene-7,8-diol
show the reaction diagram
Q9ZAG3
-
-
-
?
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(1R,2S,4R)-limonene-1,2-epoxide + H2O
(1S,2S,4R)-limonene-1,2-diol
show the reaction diagram
-
-
optically pure, diastereomeric excess above 99%
?
(1R,2S,4S)-limonene-1,2-epoxide + H2O
(1R,2R,4S)-limonene-1,2-diol
show the reaction diagram
-
-
optically pure, diastereomeric excess above 99%
?
(1S,2R,4R)-limonene-1,2-epoxide + H2O
(1S,2S,4R)-limonene-1,2-diol
show the reaction diagram
-
-
optically pure, diastereomeric excess above 99%
?
(1S2R4S)-limonene-1,2-epoxide + H2O
(1R,2R,4S)-limonene-1,2-diol
show the reaction diagram
-
-
optically pure, diastereomeric excess above 99%
?
limonene-1,2-eopxide + H2O
limonene-1,2-diol
show the reaction diagram
Rhodococcus erythropolis, Rhodococcus erythropolis DCL14
Q9ZAG3
part of limonene degradation pathway which allows the organism to grow on limone as sole source of carbon and energy
-
-
ir
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
hexanamide
-
competitive
Hexylamine
-
competitive
valpromide
-
i.e. dipropylacetamide, competitive
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1.4
-
styrene-7,8-oxide
-
pH 7.4, mutant N55A; pH 7.4, wild-type
3.7
-
styrene-7,8-oxide
-
pH 7.4, mutant Y53F
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.002
-
styrene-7,8-oxide
-
pH 7.4, mutant N55A
0.176
-
styrene-7,8-oxide
-
pH 7.4, mutant Y53F
0.47
-
styrene-7,8-oxide
-
pH 7.4, wild-type
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
2
-
hexanamide
-
pH 7.4, 1% ethanol in final assay mixture
0.035
-
Hexylamine
-
pH 7.4, 1% ethanol in final assay mixture
0.1
-
valpromide
-
pH 7.4, 1% ethanol in final assay mixture
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
85.1
-
-
with (1R,2S,4R)-limonene-1,2-epoxide as substrate
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7
-
-
very broad pH optimum
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5
10.5
-
below pH 5 chemical hydrolysis overtook biological hydrolysis rate
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
30
45
-
rapid inactivation above 45°C
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
15000
-
-
gel filtration
16520
-
-
deduced from amino acid sequence
17000
-
-
SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
dimer
-
crystallization data
monomer
-
gel filtration
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
modeling of substrate 1,2-epoxymenth-8-ene into the active site of crystal structure and evaluation of the roles of residues Arg99, Tyr53 and Asn55 by QM/MM-scannedenergy mapping
-
selenomethionine-substituted enzyme
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
49
-
-
15 min, 50% resiudal activity for wild-type
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
gel filtration, hydroxyapatite, anion exchange
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expressed in Escherichia coli
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
D101A
-
catalytically inactive, D101 is the acid catalyst that protonates the epoxide oxygen
D101N
-
catalytically inactive, D101 is the acid catalyst that protonates the epoxide oxygen
L114C/I116V
-
substrate cyclopentene-oxide, 72% conversion, (S,S)-product with 68% enantiomeric excess
L114I/I116V
-
substrate cyclopentene-oxide, 74% conversion, (S,S)-product with 50% enantiomeric excess
L114V/I116V
-
substrate cyclopentene-oxide, 72% conversion, (S,S)-product with 60% enantiomeric excess
L74I/I80C
-
substrate cyclopentene-oxide, 75% conversion, (R,R)-product with 66% enantiomeric excess
L74I/I80V
-
substrate cyclopentene-oxide, 75% conversion, (R,R)-product with 58% enantiomeric excess
L74V/I80V
-
substrate cyclopentene-oxide, 67% conversion, (R,R)-product with 53% enantiomeric excess
M32C/I80F/L114C/I116V
-
substrate rac-2-(phenoxymethyl)oxirane, 31% conversion, (2R)-product with 92% enantiomeric excess. Substrate rac-1-methyl-7-oxabicyclo[4.1.0]heptane, 99% conversion, (1S,2S)-product with 55% enantiomeric excess
M32L/L35C
-
substrate cyclopentene-oxide, 78% conversion, (S,S)-product with 16% enantiomeric excess
M32L/L35F
-
substrate cyclopentene-oxide, 79% conversion, (S,S)-product with 24% enantiomeric excess
M32L/L35V
-
substrate cyclopentene-oxide, 78% conversion, (S,S)-product with 10% enantiomeric excess
M78F/V83I
-
substrate cyclopentene-oxide, 82% conversion, (R,R)-product with 29% enantiomeric excess
M78I/V83I
-
substrate cyclopentene-oxide, 80% conversion, (R,R)-product with 13% enantiomeric excess
N55A
-
little residual activity
N55D
-
catalytically inactive
N55D/D132N
-
catalytically inactive, folding correct
R99A
-
catalytically inactive
R99H
-
catalytically inactive
R99K
-
catalytically inactive
R99Q
-
catalytically inactive
Y53F
-
partially active
L74V/I80V
Rhodococcus erythropolis DCL 14
-
substrate cyclopentene-oxide, 67% conversion, (R,R)-product with 53% enantiomeric excess
-
M32L/L35C
Rhodococcus erythropolis DCL 14
-
substrate cyclopentene-oxide, 78% conversion, (S,S)-product with 16% enantiomeric excess
-
M32L/L35F
Rhodococcus erythropolis DCL 14
-
substrate cyclopentene-oxide, 79% conversion, (S,S)-product with 24% enantiomeric excess
-
M78V/V83I
-
substrate cyclopentene-oxide, 68% conversion, (R,R)-product with 7% enantiomeric excess
additional information
-
application of directed evolution using iterative saturation mutagenesis as a means to engineer LEH mutants showing broad substrate scope with high stereoselectivity. Mutants are obtained which catalyze the desymmetrization of cyclopentene-oxide with stereoselective formation of either the (R,R)- or the (S,S)-diol on an optional basis. The mutants prove to be excellent catalysts for the desymmetrization of other meso-epoxides and for the hydrolytic kinetic resolution of racemic substrates
M32L/L35V
Rhodococcus erythropolis DCL 14
-
substrate cyclopentene-oxide, 78% conversion, (S,S)-product with 10% enantiomeric excess
-
additional information
Rhodococcus erythropolis DCL 14
-
application of directed evolution using iterative saturation mutagenesis as a means to engineer LEH mutants showing broad substrate scope with high stereoselectivity. Mutants are obtained which catalyze the desymmetrization of cyclopentene-oxide with stereoselective formation of either the (R,R)- or the (S,S)-diol on an optional basis. The mutants prove to be excellent catalysts for the desymmetrization of other meso-epoxides and for the hydrolytic kinetic resolution of racemic substrates
-
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
synthesis
-
application of directed evolution using iterative saturation mutagenesis as a means to engineer LEH mutants showing broad substrate scope with high stereoselectivity. Mutants are obtained which catalyze the desymmetrization of cyclopentene-oxide with stereoselective formation of either the (R,R)- or the (S,S)-diol on an optional basis. The mutants prove to be excellent catalysts for the desymmetrization of other meso-epoxides and for the hydrolytic kinetic resolution of racemic substrates
synthesis
Rhodococcus erythropolis DCL 14
-
application of directed evolution using iterative saturation mutagenesis as a means to engineer LEH mutants showing broad substrate scope with high stereoselectivity. Mutants are obtained which catalyze the desymmetrization of cyclopentene-oxide with stereoselective formation of either the (R,R)- or the (S,S)-diol on an optional basis. The mutants prove to be excellent catalysts for the desymmetrization of other meso-epoxides and for the hydrolytic kinetic resolution of racemic substrates
-