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Literature summary 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 .
    View publication on PubMed

Cloned(Commentary)

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

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.3.2.8 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
3.3.2.8 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

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

Inhibitors

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

Natural Substrates/ Products (Substrates)

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

Organism

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

Purification (Commentary)

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

Reaction

EC Number Reaction Comment Organism Reaction ID
3.3.2.8 1,2-epoxymenth-8-ene + H2O = menth-8-ene-1,2-diol LEH mechanism, substrate specificity and stereoselectivity Rhodococcus erythropolis
a
3.3.2.8 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.3.2.8 (1R,2S,4R)-limonene-1,2-epoxide + H2O
-
 Rhodococcus erythropolis (1R,2S,4R)-limonene-1,2-diol
-
 ?
3.3.2.8 (1R,2S,4R)-limonene-1,2-epoxide + H2O
-
 uncultured organism (1R,2S,4R)-limonene-1,2-diol
-
 ?
3.3.2.8 (1S,2R,4R)-limonene-1,2-epoxide + H2O
-
 Rhodococcus erythropolis (1S,2R,4R)-limonene-1,2-diol
-
 ?
3.3.2.8 (1S,2R,4R)-limonene-1,2-epoxide + H2O
-
 uncultured organism (1S,2R,4R)-limonene-1,2-diol
-
 ?
3.3.2.8 (4R)-limonene-1,2-epoxide + H2O
-
 Rhodococcus erythropolis (4R)-limonene-1,2-diol
-
 ?
3.3.2.8 (4R)-limonene-1,2-epoxide + H2O
-
 uncultured organism (4R)-limonene-1,2-diol
-
 ?
3.3.2.8 (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 ?
-
 ?
3.3.2.8 (4S)-limonene-1,2-epoxide + H2O
-
 Rhodococcus erythropolis (4S)-limonene-1,2-diol
-
 ?
3.3.2.8 (4S)-limonene-1,2-epoxide + H2O
-
 uncultured organism (4S)-limonene-1,2-diol
-
 ?
3.3.2.8 (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 ?
-
 ?
3.3.2.8 2-butyloxirane + H2O
-
 Rhodococcus erythropolis hexane-1,2-diol
-
 ?
3.3.2.8 2-butyloxirane + H2O
-
 uncultured organism hexane-1,2-diol
-
 ?
3.3.2.8 4-(1-methylethenyl)-cyclohexan-1,2-epoxide + H2O
-
 uncultured organism 4-(1-methylethenyl)-cyclohexan-1,2-diol
-
 ?
3.3.2.8 cycloheptene-1,2-epoxide + H2O
-
 Rhodococcus erythropolis cycloheptene-1,2-diol
-
 ?
3.3.2.8 cycloheptene-1,2-epoxide + H2O
-
 uncultured organism cycloheptene-1,2-diol
-
 ?
3.3.2.8 cyclohexene-1,2-epoxide + H2O
-
 Rhodococcus erythropolis cyclohexane-1,2-diol
-
 ?
3.3.2.8 cyclohexene-1,2-epoxide + H2O
-
 uncultured organism cyclohexane-1,2-diol
-
 ?
3.3.2.8 cyclopentene-1,2-epoxide + H2O low activity Rhodococcus erythropolis cyclopentane-1,2-diol
-
 ?
3.3.2.8 cyclopentene-1,2-epoxide + H2O low activity uncultured organism cyclopentane-1,2-diol
-
 ?
3.3.2.8 limonene-1,2-epoxide + H2O
-
 Rhodococcus erythropolis limonene-1,2-diol
-
 ?
3.3.2.8 limonene-1,2-epoxide + H2O
-
 uncultured organism limonene-1,2-diol
-
 ?
3.3.2.8 limonene-1,2-epoxide + H2O limonene-1,2-epoxide is not the natural substrate of CH55-LEH uncultured organism limonene-1,2-diol
-
 ?
3.3.2.8 limonene-1,2-epoxide + H2O limonene-1,2-epoxide is not the natural substrate of Tomsk-LEH Rhodococcus erythropolis limonene-1,2-diol
-
 ?
3.3.2.8 limonene-1,2-epoxide + H2O limonene-1,2-epoxide is not the natural substrate of Tomsk-LEH uncultured organism limonene-1,2-diol
-
 ?
3.3.2.8 additional information enzyme substrate specificity and stereospecificity, overview Rhodococcus erythropolis ?
-
 ?
3.3.2.8 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 ?
-
 ?
3.3.2.8 phenylethylenoxide + H2O
-
 Rhodococcus erythropolis 1-phenylethane-1,2-diol
-
 ?
3.3.2.8 phenylethylenoxide + H2O
-
 uncultured organism 1-phenylethane-1,2-diol
-
 ?

Subunits

EC Number Subunits Comment Organism
3.3.2.8 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
3.3.2.8 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

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

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.3.2.8 30
-
-
 Rhodococcus erythropolis
3.3.2.8 40
-
-
 uncultured organism
3.3.2.8 60
-
-
 uncultured organism

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.3.2.8 50
-
 about, TM of enzyme Re-LEH is 50°C Rhodococcus erythropolis
3.3.2.8 74.5
-
 apparent TM of enzyme Tomsk-LEH is 74.5°C uncultured organism
3.3.2.8 79.7
-
 apparent TM of enzyme CH55-LEH is 79.7°C uncultured organism

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.3.2.8 6.5
-
-
 uncultured organism
3.3.2.8 8
-
-
 uncultured organism
3.3.2.8 8
-
 assay at Rhodococcus erythropolis

General Information

EC Number General Information Comment Organism
3.3.2.8 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
3.3.2.8 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
3.3.2.8 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
3.3.2.8 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
3.3.2.8 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