Information on EC 3.3.2.8 - limonene-1,2-epoxide hydrolase and Organism(s) Rhodococcus erythropolis and UniProt Accession Q9ZAG3

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ir

(1R,2S,4R)-limonene-1,2-epoxide + H2O

(1R,2S,4R)-limonene-1,2-diol

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-

?

(1R,2S,4R)-limonene-1,2-epoxide + H2O

(1S,2S,4R)-limonene-1,2-diol

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

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ir

(1R,2S,4S)-limonene-1,2-epoxide + H2O

(1R,2R,4S)-limonene-1,2-diol

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

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ir

(1S,2R)-1-methylcyclohexane oxide + H2O

(1R,2R)-1-methylcyclohexane-1,2-diol

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ir

(1S,2R,4R)-limonene-1,2-epoxide + H2O

(1S,2R,4R)-limonene-1,2-diol

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-

?

(1S,2R,4R)-limonene-1,2-epoxide + H2O

(1S,2S,4R)-limonene-1,2-diol

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

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ir

(1S,2R,4S)-limonene-1,2-epoxide + H2O

(1R,2R,4S)-limonene-1,2-diol

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

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ir

(4R)-limonene-1,2-epoxide + H2O

(4R)-limonene-1,2-diol

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?

(4S)-limonene-1,2-epoxide + H2O

(4S)-limonene-1,2-diol

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?

1-methylcyclohexene oxide + H2O

1-methylcyclohexane-1,2-diol

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?

2 cyclohexene-1,2-epoxide + 2 H2O

(S,S)-cyclohexane-1,2-diol + (R,R)-cyclohexane-1,2-diol

LEH is the catalyst in the hydrolytic desymmetrization of cyclohexene oxide with formation of (R,R)- and (S,S)-cyclohexene-1,2-diol. Wild-type LEH shows an enanioselectivity of 2% enantiomeric excess (S,S), analysis of (R,R)- and (S,S)-selective LEH mutant variants (80-94% enantiomeric excess)

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?

2-butyloxirane + H2O

hexane-1,2-diol

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?

cycloheptene-1,2-epoxide + H2O

cycloheptene-1,2-diol

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?

cyclohexene oxide + H2O

cyclohexane-1,2-diol

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?

cyclohexene-1,2-epoxide + H2O

cyclohexane-1,2-diol

cyclopentene-1,2-epoxide + H2O

cyclopentane-1,2-diol

low activity

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?

indene oxide + H2O

indane-1,2-diol

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?

limonene-1,2-epoxide + H2O

(1S,2S,4R)-limonene-1,2-diol + (1R,2R,4S)-limonene-1,2-diol

enantiomeric mixtures of (+)-limone oxide, overview. Re-LEH catalyzes the quantitative conversion of the (+)- and (-)-limonene oxide cis/trans mixtures into the respective diols, the (+)-enantiomer is the better substrate

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?

limonene-1,2-epoxide + H2O

limonene-1,2-diol

5 entries

phenylethylenoxide + H2O

1-phenylethane-1,2-diol

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-

?

styrene-7,8-oxide + H2O

styrene-7,8-diol

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-

?

(1R,2S,4R)-limonene-1,2-epoxide + H2O

(1S,2S,4R)-limonene-1,2-diol

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optically pure, diastereomeric excess above 99%

?

(1R,2S,4S)-limonene-1,2-epoxide + H2O

(1R,2R,4S)-limonene-1,2-diol

-

optically pure, diastereomeric excess above 99%

?

(1S,2R,4R)-limonene-1,2-epoxide + H2O

(1S,2S,4R)-limonene-1,2-diol

-

optically pure, diastereomeric excess above 99%

?

(1S2R4S)-limonene-1,2-epoxide + H2O

(1R,2R,4S)-limonene-1,2-diol

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optically pure, diastereomeric excess above 99%

?

1,2-epoxy-2,6-dimethyl-5-heptene + H2O

(2S)-2,6-dimethyl-5-hepten-1,2-diol

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?

1,2-epoxy-2-methyl-6-heptene + H2O

(2S)-2-methyl-6-heptene-1,2-diol

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?

1,2-epoxy-2-methylheptane + H2O

(2S)-2-methylheptane-1,2-diol

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?

1,2-epoxy-3-benzyl-2-methylpropane + H2O

(2S)-3-benzyl-2-methylpropane-1,2-diol

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?

1-methylcyclohexene oxide + H2O

(1S,2S)-1-methylcyclohexane-1,2-diol

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?

cyclohexene oxide + H2O

(1S,2S)-trans cyclohexanediol + (1R,2R)-trans-cyclohexanediol

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?

cyclopentene-oxide + H2O

(R,R)-cyclopentene-1,2-diol + (S,S)-cyclopentene-1,2-diol

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wild-type, 72% conversion, (R,R)-product with 14% enantiomeric excess

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?

indene oxide + H2O

indane-1,2-diol

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?

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

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wild-type, 99% conversion, 19% enantiomeric excess for (1S,2S)-product

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?

rac-2-(phenoxymethyl)oxirane + H2O

(2S)-2-(phenoxymethyl)oxirane + (2R)-3-phenoxypropane-1,2-diol

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wild-type, 33% conversion, 37% enantiomeric excess

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?

additional information

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5 entries

limonene-1,2-epoxide + H2O

limonene-1,2-diol

(1R,2S,4R)-limonene-1,2-epoxide + H2O

(1S,2S,4R)-limonene-1,2-diol

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optically pure, diastereomeric excess above 99%

?

(1R,2S,4S)-limonene-1,2-epoxide + H2O

(1R,2R,4S)-limonene-1,2-diol

-

optically pure, diastereomeric excess above 99%

?

(1S,2R,4R)-limonene-1,2-epoxide + H2O

(1S,2S,4R)-limonene-1,2-diol

-

optically pure, diastereomeric excess above 99%

?

(1S2R4S)-limonene-1,2-epoxide + H2O

(1R,2R,4S)-limonene-1,2-diol

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optically pure, diastereomeric excess above 99%

?

hexanamide

competitive

Hexylamine

competitive

valpromide

6.39 - 19.41

cyclohexene-1,2-epoxide

11 entries

1.4 - 3.7

styrene-7,8-oxide

0.59 - 1.55

cyclohexene-1,2-epoxide

11 entries

0.002 - 0.47

styrene-7,8-oxide

0.042 - 0.122

cyclohexene-1,2-epoxide

11 entries

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

1.85

Re-LEH , substrate (-)-limonene oxide, pH 8.0, 20°C, non-optimized conditions

13.95

Re-LEH , substrate (+)-limonene oxide, pH 8.0, 50°C, optimized conditions

3.17

Re-LEH , substrate (-)-limonene oxide, pH 8.0, 50°C, optimized conditions

6.18

Re-LEH , substrate (+)-limonene oxide, pH 8.0, 20°C, non-optimized conditions

85.1

7.4

assay at

8

7

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very broad pH optimum

5 - 10.5

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below pH 5 chemical hydrolysis overtook biological hydrolysis rate

30

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30 - 37

assay at

50

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50

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30 - 45

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rapid inactivation above 45°C

cytosol

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-

5737

physiological function

additional information

4 entries

additional information

quantum mechanics/molecular mechanics (QM/MM) free energy calculations for the reaction with molecular dynamics simulations of the enzyme internal dynamics, and the calculation of binding affinities (using the WaterSwap method) for various representatives of the enzyme conformational ensemble, show that the presence of natural or non-natural substrates differentially modulates the dynamic and catalytic behavior of LEH. The cross-talk between the protein and the ligands favors the selection of specific substrate-dependent interactions in the binding site, priming reactive complexes to select different preferential reaction pathways. LEH substrate binding pocket structure, LEH forms a stable homodimer, and each monomer can bind a substrate molecule within its catalytic pocket, overview. Crucial role of monomer-monomer interactions in stabilizing and tuning LEH dynamics and stability. Hydrolysis by LEH occurs via a complex mechanism, the Asp101-Arg99-Asp132 triad drives a concerted reaction involving the deprotonation of a water molecule by Asp132, the nucleophilic attack of the resulting hydroxide ion on the epoxide and protonation of the oxirane ring by the protonated Asp101 (specifically labeled Ash101). Arg99 is strongly associated through hydrogen bonds and electrostatic interactions with both Asp101 and Asp132 and even if it is not directly involved in the reaction mechanism, its mutation results in a deactivated enzyme. This complex picture is completed by the proper positioning and activation of the nucleophilic water by the H-bond network formed by Asn55 and Tyr53 (and Asp132 itself). In the model, the side chains of the residues of the catalytic triad, the water molecule and the epoxide are included in the QM region. The opening of the epoxide can result from the attack on either of the two carbon atoms of the LEO oxirane ring. Experimental evidence indicates that the water molecule privileges the attack on the more substituted C1 atom

752350

Q9ZAG3 pBLAST

LIMA_RHOER

149

0

16521

Swiss-Prot

Show Sequence

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17 entries

15000

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gel filtration

16520

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deduced from amino acid sequence

208937

17000

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SDS-PAGE

dimer

4 entries

monomer

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gel filtration

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

714359

purified enzyme mutant H-2-H5 (E45D/L74F/T76K/M78F/N92K/L114V/I116V) and mutant H-2-H5 in complex with (S,S)-cyclohexenediol, sitting-drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 50 mM potassium phosphate buffer, pH 8.0, with 0.001 ml of reservoir solution containing 0.1 M HEPES, pH 7.5, 2% v/v PEG 400, and 2.0 M ammonium sulfate, and equilibration against reservoir solution, 20°C, the single protein crystals are soaked in 100 mM ligand solution for 1 min, X-ray diffraction structure determination and analysis, molecular replacement method using the wild-type LEH (PDB ID 1NU3) as a search model, comparison of crystal structures of wild-type and mutant enzymes

selenomethionine-substituted enzyme

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

N55A

N55D

catalytically inactive

N55D/D132N

catalytically inactive, folding correct

R99A

catalytically inactive

R99H

catalytically inactive

R99K

R99Q

catalytically inactive

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

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

additional information

41

wild-type LEH shows a thermostability of T50 30 (temperature at which 50% of enzyme activity is lost following a heat treatment for 30 min) = 41°C

50

about, TM of enzyme Re-LEH is 50°C

49

-

15 min, 50% resiudal activity for wild-type

additional information

R,R- and S,S-selective LEH variants (80-94% enantiomeric excess) show enhanced thermostability by 5-10°C compared to wild-type and still reasonable levels of activity

recombinant His-tagged wild-type and mutant enzymes by nickel affinity and anion exchange chromatography, followed by ultrafiltration, gel filtration, and again ultrafiltration

gel filtration, hydroxyapatite, anion exchange

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recombinant expression of His-tagged wild-type and mutant enzymes

expressed in Escherichia coli

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208937

synthesis

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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

Van der Werf, M.J.; Orru, R.V.A.; Overkamp, K.M.; Swarts, H.J.; Ospiran, I.; Steinreiber, A.; de Bont, J.A.M.; Faber, K.

Substrate specificity and stereospecificity of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14; an enzyme showing sequential and enantioconvergent substrate conversion

Appl. Microbiol. Biotechnol.

52

380-385

1999

Rhodococcus erythropolis

-

208937

Barbirato, F.; Verdoes, J.C.; de Bont, J.A.M.; van der Werf, M.J.

The Rhodococcus erythropolis DCL14 limonene-1,2-epoxide hydrolase gene encodes an enzyme belonging to a novel class of epoxide hydrolases

FEBS Lett.

438

293-296

1998

Rhodococcus erythropolis

9827564

Van der Werf, M.J.; Overkamp, K.M.; de Bont, J.A.M.

Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14 belongs to a novel class of epoxide hydrolases

J. Bacteriol.

180

5052-5057

1998

Rhodococcus erythropolis

9748436

Arand, M.; Hallberg, B.M.; Zou, J.; Bergfors, T.; Oesch, F.; Van Der Werf, M.J.; De Bont, J.A.M.; Jones, T.A.; Mowbray, S.L.

Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site

EMBO J.

22

2583-2592

2003

Rhodococcus erythropolis (Q9ZAG3), Rhodococcus erythropolis

12773375

Hopmann, K.H.; Hallberg, B.M.; Himo, F.

Catalytic mechanism of limonene epoxide hydrolase, a theoretical study

J. Am. Chem. Soc.

127

14339-14347

2005

Rhodococcus erythropolis (Q9ZAG3), Rhodococcus erythropolis DCL14 (Q9ZAG3), Rhodococcus erythropolis DCL14

16218628

714359

Hou, Q.Q.; Sheng, X.; Wang, J.H.; Liu, Y.J.; Liu, C.B.

QM/MM study of the mechanism of enzymatic limonene 1,2-epoxide hydrolysis

Biochim. Biophys. Acta

1824

263-268

2011

Rhodococcus erythropolis (Q9ZAG3), Rhodococcus erythropolis DCL14 (Q9ZAG3)

21925621

Zheng, H.; Reetz, M.T.

Manipulating the stereoselectivity of limonene epoxide hydrolase by directed evolution based on iterative saturation mutagenesis

J. Am. Chem. Soc.

132

15744-15751

2010

Rhodococcus erythropolis, Rhodococcus erythropolis DCL 14

20958062

Li, G.; Zhang, H.; Sun, Z.; Liu, X.; Reetz, M.

Multiparameter optimization in directed evolution engineering thermostability, enantioselectivity, and activity of an epoxide hydrolase

ACS Catal.

6

3679-3687

2016

Rhodococcus erythropolis (Q9ZAG3), Rhodococcus erythropolis DCL14 (Q9ZAG3)

-

752350

Rinaldi, S.; Van Der Kamp, M.; Ranaghan, K.; Mulholland, A.; Colombo, G.

Understanding complex mechanisms of enzyme reactivity the case of limonene-1,2-epoxide hydrolases

ACS Catal.

8

5698-5707

2018

Rhodococcus erythropolis (Q9ZAG3), Rhodococcus erythropolis DCL14 (Q9ZAG3)

-

Hou, Q.Q.; Sheng, X.; Wang, J.H.; Liu, Y.J.; Liu, C.B.

QM/MM study of the mechanism of enzymatic limonene 1,2-epoxide hydrolysis

Biochim. Biophys. Acta

1824

263-268

2012

Rhodococcus erythropolis (Q9ZAG3), Rhodococcus erythropolis DCL14 (Q9ZAG3)

21925621

Ferrandi, E.; Marchesi, C.; Annovazzi, C.; Riva, S.; Monti, D.; Wohlgemuth, R.

Efficient epoxide hydrolase catalyzed resolutions of (+)- and (-)-cis/trans-limonene oxides

ChemCatChem

7

3171-3178

2015

Rhodococcus erythropolis (Q9ZAG3), Rhodococcus erythropolis DCL14 (Q9ZAG3), uncultured organism (A0A0G3IAY2), uncultured organism (A0A0G3ICV8)

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