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Literature summary extracted from

  • Blank, P.N.; Barrow, G.H.; Chou, W.K.W.; Duan, L.; Cane, D.E.; Christianson, D.W.
    Substitution of aromatic residues with polar residues in the active site pocket of epi-isozizaene synthase leads to the generation of new cyclic sesquiterpenes (2017), Biochemistry, 56, 5798-5811 .
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.2.3.37 purified wild-type enzyme and mutant F95N in complex with Mg2+, benzyl triethylammonium cation (BTAC), and diphosphate, sitting drop vapor diffusion method, mixing of 5 mm nl of 5 mg/ml protein in 300 mM NaCl, 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM tris(2-carboxyethyl)phosphine (TCEP), 10% glycerol, 2 mM sodium pyrophosphate, and 2 mM BTAC with 600 nl of precipitant solution containing 0.17 M sodium acetate trihydrate, 85 mM sodium cacodylate trihydrate, pH 6.5, 25.5% PEG 8000, and 15% glycerol, addition of 100 nL of the 100fold dilution of wild-type enzyme crystallization seed stock, equilibration against a 0.1 ml reservoir of the precipitant solution at room temperature, 6 days, X-ray diffraction structure determination and analysis at 1.8 A and 2.51 A resolution, respectively Streptomyces coelicolor

Protein Variants

EC Number Protein Variants Comment Organism
4.2.3.37 F95C site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor
4.2.3.37 F95N site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor
4.2.3.37 F95Q site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor
4.2.3.37 F95Y site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor
4.2.3.37 F96H site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor
4.2.3.37 F96M site-directed mutagenesis, the substitution converts the epi-isozizaene synthase into a high-fidelity sesquisabinene synthase, the mutant generates 91% sesquisabinene A Streptomyces coelicolor
4.2.3.37 F96N site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor
4.2.3.37 F96Q site-directed mutagenesis, the substitution converts the epi-isozizaene synthase into a high-fidelity sesquisabinene synthase, the mutant generates 97% sesquisabinene A Streptomyces coelicolor
4.2.3.37 F96S site-directed mutagenesis, the substitution converts the epi-isozizaene synthase into a high-fidelity sesquisabinene synthase, the mutant generates 78% sesquisabinene A Streptomyces coelicolor
4.2.3.37 F96T site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor
4.2.3.37 additional information residues defining the three-dimensional contour of the hydrophobic pocket can be substituted without triggering significant structural changes elsewhere in the active site. More radical nonpolar-polar amino acid substitutions are considered when terpenoid cyclase active sites are remolded by mutagenesis with the goal of exploring and expanding product chemodiversity. Substitution of hydrophobic residues with other hydrophobic residues remolds the template and expands product chemodiversity. The substitution of hydrophobic residues, specifically, Y69, F95, F96, and W203, with polar side chains also yields functional enzyme catalysts that expand product chemodiversity. The substitution of polar residues for F96 yields high-fidelity sesquisabinene synthases. Proposed reaction mechanisms of FPP cyclization leading to products identified by GC-MS analysis catalyzed by wild-type and mutant EIZS enzymes, overview Streptomyces coelicolor
4.2.3.37 W203H site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor
4.2.3.37 W203Y site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor
4.2.3.37 Y69A site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor
4.2.3.37 Y69F site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type Streptomyces coelicolor

Inhibitors

EC Number Inhibitors Comment Organism Structure
4.2.3.37 EDTA complete inhibition Streptomyces coelicolor

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.3.37 Mg2+ required Streptomyces coelicolor

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.3.37 (2E,6E)-farnesyl diphosphate Streptomyces coelicolor
-
 (+)-epi-isozizaene + diphosphate
-
 ?
4.2.3.37 (2E,6E)-farnesyl diphosphate Streptomyces coelicolor A3(2)
-
 (+)-epi-isozizaene + diphosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
4.2.3.37 Streptomyces coelicolor Q9K498
-
-
4.2.3.37 Streptomyces coelicolor A3(2) Q9K498
-
-

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
4.2.3.37 4
-
 recombinant mutants F96S and F96N, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 4.2
-
 recombinant mutant W230H, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 5
-
 recombinant mutants F95N and Y69A, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 5.5
-
 recombinant mutant F95C, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 5.8
-
 recombinant mutant F96H, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 5.9
-
 recombinant mutant F96T, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 5.92
-
 recombinant mutant F95Q, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 6
-
 recombinant mutant W230Y, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 7
-
 recombinant mutant F96M, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 7.6
-
 recombinant mutant Y69F, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 8
-
 recombinant mutants F96Q and F95Y, pH 7.5, 30°C Streptomyces coelicolor
4.2.3.37 18
-
 recombinant wild-type enzyme, pH 7.5, 30°C Streptomyces coelicolor

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.3.37 (2E,6E)-farnesyl diphosphate
-
 Streptomyces coelicolor (+)-epi-isozizaene + diphosphate
-
 ?
4.2.3.37 (2E,6E)-farnesyl diphosphate
-
 Streptomyces coelicolor A3(2) (+)-epi-isozizaene + diphosphate
-
 ?

Synonyms

EC Number Synonyms Comment Organism
4.2.3.37 EIZS
-
 Streptomyces coelicolor
4.2.3.37 epi-isozizaene synthase
-
 Streptomyces coelicolor

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.3.37 30
-
 assay at Streptomyces coelicolor

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.3.37 7.5
-
 assay at Streptomyces coelicolor

General Information

EC Number General Information Comment Organism
4.2.3.37 malfunction substitution of hydrophobic residues with other hydrophobic residues remolds the template and expands product chemodiversity. The substitution of hydrophobic residues, specifically, Y69, F95, F96, and W203, with polar side chains also yields functional enzyme catalysts that expand product chemodiversity. The substitution of polar residues for F96 yields high-fidelity sesquisabinene synthases. Residues defining the three-dimensional contour of the hydrophobic pocket can be substituted without triggering significant structural changes elsewhere in the active site Streptomyces coelicolor
4.2.3.37 physiological function the sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic hydrocarbon precursor of the antibiotic albaflavenone. The hydrophobic active site pocket of EIZS serves as a template as it binds and chaperones the flexible substrate and carbocation intermediates through the conformations required for a multistep reaction sequence Streptomyces coelicolor