3.3.2.8: limonene-1,2-epoxide hydrolase
This is an abbreviated version!
For detailed information about limonene-1,2-epoxide hydrolase, go to the full flat file.
Word Map on EC 3.3.2.8
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3.3.2.8
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stereoselective
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rhodococcus
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erythropolis
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hydrolases
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desymmetrization
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cyclohexene
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enantioselective
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alphabet
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regioselective
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lining
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six-stranded
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astronomically
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intricacies
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valpromide
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selenomethionine-substituted
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biocatalysis
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single-wavelength
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wide-ranging
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diols
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polyketide
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cyclopentene
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synthesis
- 3.3.2.8
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stereoselective
- rhodococcus
- erythropolis
- hydrolases
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desymmetrization
- cyclohexene
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enantioselective
-
alphabet
-
regioselective
-
lining
-
six-stranded
-
astronomically
-
intricacies
- valpromide
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selenomethionine-substituted
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biocatalysis
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single-wavelength
-
wide-ranging
- diols
- polyketide
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cyclopentene
- synthesis
Reaction
Synonyms
CH55-LEH, LEH, limA, limonene 1,2-epoxide hydrolase, limonene epoxide hydrolase, limonene oxide hydrolase, limonene-1,2-epoxide hydrolase, Re-LEH, Tomsk-LEH
ECTree
3.3.2.8 limonene-1,2-epoxide hydrolaseAdvanced search results
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results (Engineering
Engineering on EC 3.3.2.8 - limonene-1,2-epoxide hydrolase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
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
E45D/L74F/T76K/M78F/N92K/L114V/I116V
site-directed mutagenesis, the mutant lacking the N- and C-terminal mutations displays 86% enantiomeric excess in favor of (S,S)-cyclohexene-1,2-diol
I5C/S15P/A19K/T76K/E84C/T85V/G89C/S91C/N92K/Y96F/E124D
site-directed mutagenesis, the multisite mutant shows enhanced and inverted enantioselectivity, and an increase in apparent melting temperature relative to wild-type LEH from 50 to 85°C and a more than 250fold longer half-life
L114C/I116V
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substrate cyclopentene-oxide, 72% conversion, (S,S)-product with 68% enantiomeric excess
L114I/I116V
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substrate cyclopentene-oxide, 74% conversion, (S,S)-product with 50% enantiomeric excess
L114V/I116V
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substrate cyclopentene-oxide, 72% conversion, (S,S)-product with 60% enantiomeric excess
L74I/I80C
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substrate cyclopentene-oxide, 75% conversion, (R,R)-product with 66% enantiomeric excess
L74I/I80V
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substrate cyclopentene-oxide, 75% conversion, (R,R)-product with 58% enantiomeric excess
L74V/I80V
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substrate cyclopentene-oxide, 67% conversion, (R,R)-product with 53% enantiomeric excess
M32C/I80F/L114C/I116V
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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
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substrate cyclopentene-oxide, 78% conversion, (S,S)-product with 16% enantiomeric excess
M32L/L35F
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substrate cyclopentene-oxide, 79% conversion, (S,S)-product with 24% enantiomeric excess
M32L/L35V
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substrate cyclopentene-oxide, 78% conversion, (S,S)-product with 10% enantiomeric excess
M78F/V83I
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substrate cyclopentene-oxide, 82% conversion, (R,R)-product with 29% enantiomeric excess
M78I/V83I
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substrate cyclopentene-oxide, 80% conversion, (R,R)-product with 13% enantiomeric excess
M78V/V83I
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substrate cyclopentene-oxide, 68% conversion, (R,R)-product with 7% enantiomeric excess
N55A
R99K
S15P/A19K/E45K/T76K/T85V/N92K/Y96F/E124D
site-directed mutagenesis, the multisite mutant shows enhanced and inverted enantioselectivity, and an increase in apparent melting temperature relative to wild-type LEH from 50 to 85°C and a more than 250fold longer half-life, T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) is 46°C and the enantiomeric excess is 80% in favor of (R,R)-cyclohexene-1,2-diol
S15P/M78F
site-directed mutagenesis, T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) is 47°C and the enantiomeric excess is 34% in favor of (R,R)-cyclohexene-1,2-diol
S15P/M78F/N92K/F139V
site-directed mutagenesis, T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) is 48°C and the enantiomeric excess is 39% in favor of (R,R)-cyclohexene-1,2-diol
S15P/M78F/N92K/F139V/T76K/T85K
site-directed mutagenesis, T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) is 44°C and the enantiomeric excess is 45% in favor of (R,R)-cyclohexene-1,2-diol
S15P/M78F/N92K/F139V/T76K/T85K/E45D/I80V/E124D
site-directed mutagenesis, T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) is 46°C and the enantiomeric excess is 80% in favor of (R,R)-cyclohexene-1,2-diol
T76D/L114V/I116V
site-directed mutagenesis, the mutant lacking the N- and C-terminal mutations, maintains enantioselectivity of 71% enantiomeric excess in favor of (S,S)-cyclohexene-1,2-diol
T76K/L114V/I116V
site-directed mutagenesis, the mutant lacking the N- and C-terminal mutations, maintains enantioselectivity of 71% enantiomeric excess in favor of (S,S)-cyclohexene-1,2-diol, T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) is 44°C
T76K/L114V/I116V/F139V/L147F
site-directed mutagenesis, the mutant lacking the N-terminal mutations shows enantioselectivities of 82% enantiomeric excess in favor of (S,S)-cyclohexene-1,2-diol, T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) is 45°C
T76K/L114V/I116V/N92D/F139V/L147F
site-directed mutagenesis, the mutant lacking the N-terminal mutations shows enantioselectivities of 82% enantiomeric excess in favor of (S,S)-cyclohexene-1,2-diol
T76K/L114V/I116V/N92K/F139V/L147F
site-directed mutagenesis, the mutant lacking the N-terminal mutations shows enantioselectivities of 83% enantiomeric excess in favor of (S,S)-cyclohexene-1,2-diol
T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) is 51°C, and the enantiomeric excess is 92% in favor of (S,S)-cyclohexene-1,2-diol
T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F/E45D
site-directed mutagenesis, T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) is 51°C and the enantiomeric excess is 94% in favor of (S,S)-cyclohexene-1,2-diol
Y53F
L74V/I80V
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substrate cyclopentene-oxide, 67% conversion, (R,R)-product with 53% enantiomeric excess
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M32L/L35C
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substrate cyclopentene-oxide, 78% conversion, (S,S)-product with 16% enantiomeric excess
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M32L/L35F
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substrate cyclopentene-oxide, 79% conversion, (S,S)-product with 24% enantiomeric excess
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M32L/L35V
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substrate cyclopentene-oxide, 78% conversion, (S,S)-product with 10% enantiomeric excess
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E45D/L74F/T76K/M78F/N92K/L114V/I116V
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site-directed mutagenesis, the mutant lacking the N- and C-terminal mutations displays 86% enantiomeric excess in favor of (S,S)-cyclohexene-1,2-diol
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I5C/S15P/A19K/T76K/E84C/T85V/G89C/S91C/N92K/Y96F/E124D
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site-directed mutagenesis, the multisite mutant shows enhanced and inverted enantioselectivity, and an increase in apparent melting temperature relative to wild-type LEH from 50 to 85°C and a more than 250fold longer half-life
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N55A
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site-directed mutagenesis, QM/MM-optimized active structure of the enzyme mutant compared to wild-type
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R99K
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site-directed mutagenesis, QM/MM-optimized active structure of the enzyme mutant compared to wild-type
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S15P/A19K/E45K/T76K/T85V/N92K/Y96F/E124D
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site-directed mutagenesis, the multisite mutant shows enhanced and inverted enantioselectivity, and an increase in apparent melting temperature relative to wild-type LEH from 50 to 85°C and a more than 250fold longer half-life, T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) is 46°C and the enantiomeric excess is 80% in favor of (R,R)-cyclohexene-1,2-diol
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T76K/L114V/I116V/N92K/F139V/L147F
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site-directed mutagenesis, the mutant lacking the N-terminal mutations shows enantioselectivities of 83% enantiomeric excess in favor of (S,S)-cyclohexene-1,2-diol
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Y53F
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site-directed mutagenesis, QM/MM-optimized active structure of the enzyme mutant compared to wild-type
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D80A
site-directed mutagenesis, mutation of the catalytic residue, inactive mutant
D82A
site-directed mutagenesis, mutation of the catalytic residue, inactive mutant
I80Y/L114V/I116V
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site-directed mutagenesis, mutant SZ348, no or poor activity with cyclohexene-1,2-epoxide
L74F/M78F/I80F/L114V/I116V/F139V
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site-directed mutagenesis, mutant SZ719
L74F/M78F/L103V/L114V/I116V/F139V/L147V
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site-directed mutagenesis, mutant SZ92
L74F/M78V/I80V/L114F
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site-directed mutagenesis, mutant SZ338, no activity with cyclohexene-1,2-epoxide
M32V/M78V/I80V/L114FM32V/M78V/I80V/L114F
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site-directed mutagenesis, mutant SZ529
additional information
N55A
site-directed mutagenesis, QM/MM-optimized active structure of the enzyme mutant compared to wild-type
R99K
site-directed mutagenesis, QM/MM-optimized active structure of the enzyme mutant compared to wild-type
Y53F
site-directed mutagenesis, QM/MM-optimized active structure of the enzyme mutant compared to wild-type
additional information
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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
additional information
comparison of preparative resolution of (+)- and (-)-limonene oxide mixtures catalyzed by different LEHs under non-optimized conditions, optimization of the biocatalyzed processes to obtain enantiomerically pure isomers, overview
additional information
mutations E45D, T76K, and N92K are located on or near the surface of LEH. It is likely that these mutations stabilize the protein by optimizing the distribution of charges on the enzyme surface, which is an established method of protein stabilization. Furthermore, S15D may form an ionic bond with A19K, thereby stabilizing a flexible N-terminal loop. Evolved thermostability-related mutations, structure-function relationships, overview
additional information
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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
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additional information
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mutations E45D, T76K, and N92K are located on or near the surface of LEH. It is likely that these mutations stabilize the protein by optimizing the distribution of charges on the enzyme surface, which is an established method of protein stabilization. Furthermore, S15D may form an ionic bond with A19K, thereby stabilizing a flexible N-terminal loop. Evolved thermostability-related mutations, structure-function relationships, overview
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additional information
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comparison of preparative resolution of (+)- and (-)-limonene oxide mixtures catalyzed by different LEHs under non-optimized conditions, optimization of the biocatalyzed processes to obtain enantiomerically pure isomers, overview
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additional information
comparison of preparative resolution of (+)- and (-)-limonene oxide mixtures catalyzed by different LEHs under non-optimized conditions, optimization of the biocatalyzed processes, overview. The obtainment of the cis isomer from the same (+)-limonene oxide mixture by Tomsk-LEH-catalyzed resolution is not significantly improved
additional information
comparison of preparative resolution of (+)- and (-)-limonene oxide mixtures catalyzed by different LEHs under non-optimized conditions, optimization of the biocatalyzed processes, overview. The obtainment of the cis isomer from the same (+)-limonene oxide mixture by Tomsk-LEH-catalyzed resolution is not significantly improved
additional information
comparison of preparative resolution of (+)- and (-)-limonene oxide mixtures catalyzed by different LEHs under non-optimized conditions, optimization of the biocatalyzed processes, overview. The obtainment of the enantiomerically pure trans isomer, the increase of the reaction temperature to 50°C leads to an excellent resolution even at a substrate loading of 2 mol/l in reasonable reaction times
additional information
comparison of preparative resolution of (+)- and (-)-limonene oxide mixtures catalyzed by different LEHs under non-optimized conditions, optimization of the biocatalyzed processes, overview. The obtainment of the enantiomerically pure trans isomer, the increase of the reaction temperature to 50°C leads to an excellent resolution even at a substrate loading of 2 mol/l in reasonable reaction times
additional information
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structural and computational insight into the catalytic mechanism of limonene epoxide hydrolase mutants in stereoselective transformations
