Literature summary for 4.2.3.37 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 .

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
F95C	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor
F95N	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor
F95Q	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor
F95Y	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor
F96H	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor
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
F96N	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor
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
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
F96T	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor
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
W203H	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor
W203Y	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor
Y69A	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor
Y69F	site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type	Streptomyces coelicolor

Inhibitors

Inhibitors	Comment	Organism	Structure
EDTA	complete inhibition	Streptomyces coelicolor

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Streptomyces coelicolor

Natural Substrates/ Products (Substrates)

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

Organism

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

Specific Activity [micromol/min/mg]

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

Substrates and Products (Substrate)

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

Synonyms

Synonyms	Comment	Organism
EIZS	-	Streptomyces coelicolor
epi-isozizaene synthase	-	Streptomyces coelicolor

Temperature Optimum [°C]

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

pH Optimum

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

General Information

General Information	Comment	Organism
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
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