Literature summary for 3.3.2.8 extracted from

Li, G.; Zhang, H.; Sun, Z.; Liu, X.; Reetz, M.
Multiparameter optimization in directed evolution engineering thermostability, enantioselectivity, and activity of an epoxide hydrolase (2016), ACS Catal., 6, 3679-3687 .

No PubMed abstract available

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of His-tagged wild-type and mutant enzymes	Rhodococcus erythropolis

Crystallization (Commentary)

Crystallization (Comment)	Organism
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	Rhodococcus erythropolis

Crystallization (Comment)

Organism

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

Rhodococcus erythropolis

Protein Variants

Protein Variants	Comment	Organism
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
6.39	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F/N92K/F139V/T76K/T85K/E45D/I80V/E124D	Rhodococcus erythropolis
6.7	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant wild-type enzyme	Rhodococcus erythropolis
6.96	-	cyclohexene-1,2-epoxide	pH 7.4, 37°C, recombinant mutant S15P/M78F/N92K/F139V/T76K/T85K/E45D/I80V/E124D	Rhodococcus erythropolis
8.23	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F/N92K/F139V/T76K/T85K	Rhodococcus erythropolis
13.09	-	cyclohexene-1,2-epoxide	pH 7.4, 37°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F/E45D	Rhodococcus erythropolis
14.41	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V	Rhodococcus erythropolis
15.44	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F	Rhodococcus erythropolis
16.06	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F/N92K/F139V	Rhodococcus erythropolis
16.1	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F/E45D	Rhodococcus erythropolis
16.97	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F	Rhodococcus erythropolis
19.41	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F	Rhodococcus erythropolis

Organism

Organism	UniProt	Comment	Textmining
Rhodococcus erythropolis	Q9ZAG3	-	-
Rhodococcus erythropolis DCL14	Q9ZAG3	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes by nickel affinity and anion exchange chromatography, followed by ultrafiltration, gel filtration, and again ultrafiltration	Rhodococcus erythropolis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2 cyclohexene-1,2-epoxide + 2 H2O	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)	Rhodococcus erythropolis	(S,S)-cyclohexane-1,2-diol + (R,R)-cyclohexane-1,2-diol	-	?
2 cyclohexene-1,2-epoxide + 2 H2O	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)	Rhodococcus erythropolis DCL14	(S,S)-cyclohexane-1,2-diol + (R,R)-cyclohexane-1,2-diol	-	?
additional information	enantioselectivity and activity of limonene epoxide hydrolase, overview	Rhodococcus erythropolis	?	-	?
additional information	enantioselectivity and activity of limonene epoxide hydrolase, overview	Rhodococcus erythropolis DCL14	?	-	?

Synonyms

Synonyms	Comment	Organism
LEH	-	Rhodococcus erythropolis
limonene epoxide hydrolase	-	Rhodococcus erythropolis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	37	assay at	Rhodococcus erythropolis

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
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	Rhodococcus erythropolis
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	Rhodococcus erythropolis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.59	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F/N92K/F139V/T76K/T85K	Rhodococcus erythropolis
0.64	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F/N92K/F139V/T76K/T85K/E45D/I80V/E124D	Rhodococcus erythropolis
0.76	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F	Rhodococcus erythropolis
0.82	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant wild-type enzyme	Rhodococcus erythropolis
0.82	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F	Rhodococcus erythropolis
0.84	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F/E45D	Rhodococcus erythropolis
0.84	-	cyclohexene-1,2-epoxide	pH 7.4, 37°C, recombinant mutant S15P/M78F/N92K/F139V/T76K/T85K/E45D/I80V/E124D	Rhodococcus erythropolis
0.86	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F/N92K/F139V	Rhodococcus erythropolis
0.93	-	cyclohexene-1,2-epoxide	pH 7.4, 37°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F/E45D	Rhodococcus erythropolis
1.25	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F	Rhodococcus erythropolis
1.55	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V	Rhodococcus erythropolis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.4	-	assay at	Rhodococcus erythropolis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.042	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F	Rhodococcus erythropolis
0.049	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F	Rhodococcus erythropolis
0.052	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F/E45D	Rhodococcus erythropolis
0.054	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F/N92K/F139V	Rhodococcus erythropolis
0.071	-	cyclohexene-1,2-epoxide	pH 7.4, 37°C, recombinant mutant T76K/L114V/I116V/N92K/F139V/L147F/S15D/A19K/L74F/M78F/E45D	Rhodococcus erythropolis
0.072	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F/N92K/F139V/T76K/T85K	Rhodococcus erythropolis
0.074	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F	Rhodococcus erythropolis
0.1	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant S15P/M78F/N92K/F139V/T76K/T85K/E45D/I80V/E124D	Rhodococcus erythropolis
0.108	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant mutant T76K/L114V/I116V	Rhodococcus erythropolis
0.121	-	cyclohexene-1,2-epoxide	pH 7.4, 37°C, recombinant mutant S15P/M78F/N92K/F139V/T76K/T85K/E45D/I80V/E124D	Rhodococcus erythropolis
0.122	-	cyclohexene-1,2-epoxide	pH 7.4, 30°C, recombinant wild-type enzyme	Rhodococcus erythropolis