Literature summary for 3.3.2.8 extracted from

Ferrandi, E.E.; Sayer, C.; Isupov, M.N.; Annovazzi, C.; Marchesi, C.; Iacobone, G.; Peng, X.; Bonch-Osmolovskaya, E.; Wohlgemuth, R.; Littlechild, J.A.; Monti, D.
Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries (2015), FEBS J., 282, 2879-2894 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of C-terminally His6-tagged enzyme in Escherichia coli strain 10G	uncultured organism

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme in apoform or in complex with inhibitor poly(ethylene glycol), 10 mg/ml protein solution is mixed with an equal volume of precipitant solution and is covered with a 1:1 mix of silicon and paraffin oils, X-ray diffraction structure determination and analysis at 1.42-1.47 A resolution	uncultured organism
purified recombinant enzyme in apoform or in complex with inhibitor valpromide, 10 mg/ml protein solution is mixed with an equal volume of precipitant solution and is covered with a 1:1 mix of silicon and paraffin oils, X-ray diffraction structure determination and analysis at 1.16-1.26 A resolution	uncultured organism

Protein Variants

Protein Variants	Comment	Organism
D80A	site-directed mutagenesis, mutation of the catalytic residue, inactive mutant	uncultured organism
D82A	site-directed mutagenesis, mutation of the catalytic residue, inactive mutant	uncultured organism

Inhibitors

Inhibitors	Comment	Organism
poly(ethylene glycol)	enzyme binding structure, overview	uncultured organism
valpromide	binds at the active site, enzyme binding structure, overview	Rhodococcus erythropolis
valpromide	enzyme binding structure, overview	uncultured organism

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
limonene-1,2-epoxide + H2O	Rhodococcus erythropolis	-	limonene-1,2-diol	-	?
limonene-1,2-epoxide + H2O	uncultured organism	-	limonene-1,2-diol	-	?

Organism

Organism	UniProt	Comment	Textmining
Rhodococcus erythropolis	Q9ZAG3	-	-
uncultured organism	A0A0G3IAY2	-	-
uncultured organism	A0A0G3ICV8	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant soluble C-terminally His6-tagged enzyme from Escherichia coli strain 10G	uncultured organism

Reaction

Reaction	Comment	Organism	Reaction ID
1,2-epoxymenth-8-ene + H2O = menth-8-ene-1,2-diol	LEH mechanism, substrate specificity and stereoselectivity	Rhodococcus erythropolis	a
1,2-epoxymenth-8-ene + H2O = menth-8-ene-1,2-diol	LEH mechanism, substrate specificity and stereoselectivity	uncultured organism	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(1R,2S,4R)-limonene-1,2-epoxide + H2O	-	Rhodococcus erythropolis	(1R,2S,4R)-limonene-1,2-diol	-	?
(1R,2S,4R)-limonene-1,2-epoxide + H2O	-	uncultured organism	(1R,2S,4R)-limonene-1,2-diol	-	?
(1S,2R,4R)-limonene-1,2-epoxide + H2O	-	Rhodococcus erythropolis	(1S,2R,4R)-limonene-1,2-diol	-	?
(1S,2R,4R)-limonene-1,2-epoxide + H2O	-	uncultured organism	(1S,2R,4R)-limonene-1,2-diol	-	?
(4R)-limonene-1,2-epoxide + H2O	-	Rhodococcus erythropolis	(4R)-limonene-1,2-diol	-	?
(4R)-limonene-1,2-epoxide + H2O	-	uncultured organism	(4R)-limonene-1,2-diol	-	?
(4R)-limonene-1,2-epoxide + H2O	the mixture of cis (1R,2S,4R) and trans (1S,2R,4R) isomers of (+)-limonene-1,2-epoxide and the mixture of cis (1S,2R,4S) and trans (1R,2S,4S) isomers of (-)-limonene-1,2-epoxide are quantitatively converted into the diaxial (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diols, respectively. Cyclopentene-1,2-epoxide is no substrate for enzyme CH55-LEH. Enzyme substrate specificity and stereospecificity, overview	uncultured organism	?	-	?
(4S)-limonene-1,2-epoxide + H2O	-	Rhodococcus erythropolis	(4S)-limonene-1,2-diol	-	?
(4S)-limonene-1,2-epoxide + H2O	-	uncultured organism	(4S)-limonene-1,2-diol	-	?
(4S)-limonene-1,2-epoxide + H2O	the mixture of cis (1R,2S,4R) and trans (1S,2R,4R) isomers of (+)-limonene-1,2-epoxide and the mixture of cis (1S,2R,4S) and trans (1R,2S,4S) isomers of (-)-limonene-1,2-epoxide are quantitatively converted into the diaxial (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diols, respectively. Cyclopentene-1,2-epoxide is no substrate for enzyme CH55-LEH. Enzyme substrate specificity and stereospecificity, overview	uncultured organism	?	-	?
2-butyloxirane + H2O	-	Rhodococcus erythropolis	hexane-1,2-diol	-	?
2-butyloxirane + H2O	-	uncultured organism	hexane-1,2-diol	-	?
4-(1-methylethenyl)-cyclohexan-1,2-epoxide + H2O	-	uncultured organism	4-(1-methylethenyl)-cyclohexan-1,2-diol	-	?
cycloheptene-1,2-epoxide + H2O	-	Rhodococcus erythropolis	cycloheptene-1,2-diol	-	?
cycloheptene-1,2-epoxide + H2O	-	uncultured organism	cycloheptene-1,2-diol	-	?
cyclohexene-1,2-epoxide + H2O	-	Rhodococcus erythropolis	cyclohexane-1,2-diol	-	?
cyclohexene-1,2-epoxide + H2O	-	uncultured organism	cyclohexane-1,2-diol	-	?
cyclopentene-1,2-epoxide + H2O	low activity	Rhodococcus erythropolis	cyclopentane-1,2-diol	-	?
cyclopentene-1,2-epoxide + H2O	low activity	uncultured organism	cyclopentane-1,2-diol	-	?
limonene-1,2-epoxide + H2O	-	Rhodococcus erythropolis	limonene-1,2-diol	-	?
limonene-1,2-epoxide + H2O	-	uncultured organism	limonene-1,2-diol	-	?
limonene-1,2-epoxide + H2O	limonene-1,2-epoxide is not the natural substrate of CH55-LEH	uncultured organism	limonene-1,2-diol	-	?
limonene-1,2-epoxide + H2O	limonene-1,2-epoxide is not the natural substrate of Tomsk-LEH	Rhodococcus erythropolis	limonene-1,2-diol	-	?
limonene-1,2-epoxide + H2O	limonene-1,2-epoxide is not the natural substrate of Tomsk-LEH	uncultured organism	limonene-1,2-diol	-	?
additional information	enzyme substrate specificity and stereospecificity, overview	Rhodococcus erythropolis	?	-	?
additional information	the mixture of cis (1R,2S,4R) and trans (1S,2R,4R) isomers of (+)-limonene-1,2-epoxide and the mixture of cis (1S,2R,4S) and trans (1R,2S,4S) isomers of (-)-limonene-1,2-epoxide are quantitatively converted into the diaxial (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diols, respectively. Enzyme substrate specificity and stereospecificity, overview	uncultured organism	?	-	?
phenylethylenoxide + H2O	-	Rhodococcus erythropolis	1-phenylethane-1,2-diol	-	?
phenylethylenoxide + H2O	-	uncultured organism	1-phenylethane-1,2-diol	-	?

Subunits

Subunits	Comment	Organism
dimer	the LEH monomer fold contains a curved six-stranded mixed beta-sheet, with three alpha-helices packed onto its concave side to form the active site pocket	Rhodococcus erythropolis
dimer	the LEH monomer fold contains a curved six-stranded mixed beta-sheet, with three alpha-helices packed onto its concave side to form the active site pocket	uncultured organism

Synonyms

Synonyms	Comment	Organism
CH55-LEH	-	uncultured organism
LEH	-	Rhodococcus erythropolis
LEH	-	uncultured organism
limA	-	Rhodococcus erythropolis
limA	-	uncultured organism
Re-LEH	-	Rhodococcus erythropolis
Tomsk-LEH	-	uncultured organism

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	-	Rhodococcus erythropolis
40	-	-	uncultured organism
60	-	-	uncultured organism

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
50	-	about, TM of enzyme Re-LEH is 50°C	Rhodococcus erythropolis
74.5	-	apparent TM of enzyme Tomsk-LEH is 74.5°C	uncultured organism
79.7	-	apparent TM of enzyme CH55-LEH is 79.7°C	uncultured organism

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6.5	-	-	uncultured organism
8	-	-	uncultured organism
8	-	assay at	Rhodococcus erythropolis

General Information

General Information	Comment	Organism
malfunction	replacement of th catalytic aspartic acid residue to alanine (D82A) completely abolishes activity towards the tested substrates cyclohexene epoxide and (+)-limonene epoxide	uncultured organism
malfunction	replacement of the catalytic aspartic acid residue to alanine (D80A) completely abolishes activity towards the tested substrates cyclohexene epoxide and (+)-limonene epoxide	uncultured organism
additional information	the enzyme's catalytic mechanism is different from that of the epoxide hydrolases, EHs, belonging to the alpha/beta-hydrolase superfamily. The LEH enzyme active site contains three residues (Asp101, Arg99, and Asp132) that act in a concerted fashion to activate a water molecule which is able to open the epoxide ring without the formation of a covalently bound alkyl-enzyme intermediate. Importance of the catalytic Asp80 residues for the enzymatic activity of Tomsk-LEH. The LEH substrate binding pocket appears to have high affinity for polar molecules and additional electron density is observed in the active site pocket in the different LEH structure. Active site structure, overview	uncultured organism
additional information	the enzyme's catalytic mechanism is different from that of the epoxide hydrolases, EHs, belonging to the alpha/beta-hydrolase superfamily. The LEH enzyme active site contains three residues (Asp101, Arg99, and Asp132) that act in a concerted fashion to activate a water molecule which is able to open the epoxide ring without the formation of a covalently bound alkyl-enzyme intermediate. Importance of the catalytic Asp82 residues for the enzymatic activity of CH55-LEH. The LEH substrate binding pocket appears to have high affinity for polar molecules and additional electron density is observed in the active site pocket in the different LEH structure. Active site structure, overview	uncultured organism
additional information	the N-terminal extension of Re-LEH involved in the intersubunit interface which increases the buried surface area. The LEH monomer fold contains a curved six-stranded mixed beta-sheet, with three alpha-helices packed onto its concave side to form the active site pocket. Active site structure, overview	Rhodococcus erythropolis