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Information on EC 4.2.3.37 - epi-isozizaene synthase and Organism(s) Streptomyces coelicolor and UniProt Accession Q9K499

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EC Tree
     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.3 Acting on phosphates
                4.2.3.37 epi-isozizaene synthase
IUBMB Comments
Requires Mg2+ for activity. The displacement of the diphosphate group of farnesyl diphosphate occurs with retention of configuration . In the soil-dwelling bacterium Streptomyces coelicolor A3(2), the product of this reaction is used by EC 1.14.13.106, epi-isozizaene 5-monooxygenase, to produce the sesquiterpene antibiotic albaflavenone .
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Streptomyces coelicolor
UNIPROT: Q9K499
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The taxonomic range for the selected organisms is: Streptomyces coelicolor
The enzyme appears in selected viruses and cellular organisms
Synonyms
epi-isozizaene synthase, sav_3032, sco5222 protein, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
epi-isozizaene synthase
-
epi-isozizaene synthase
-
SCO5222 protein
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(2E,6E)-farnesyl diphosphate = (+)-epi-isozizaene + diphosphate
show the reaction diagram
ionization and isomerization of farnesyl diphosphate will give (3R)-nerolidyl diphosphate. Rotation about the newly generated C-2/C-3 bond generates the corresponding cisoid nerolidyl diphosphate conformer which can undergo ionization and cyclization to form a bisabolyl cation. Following 1,2-hydride shift and spirocyclization, the resultant acorenyl cation can undergo further cyclization, ring contraction, methyl migration, and deprotonation to yield (+)-epi-isozizaene
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SYSTEMATIC NAME
IUBMB Comments
(2E,6E)-farnesyl-diphosphate diphosphate-lyase [(+)-epi-isozizaene-forming]
Requires Mg2+ for activity. The displacement of the diphosphate group of farnesyl diphosphate occurs with retention of configuration [1]. In the soil-dwelling bacterium Streptomyces coelicolor A3(2), the product of this reaction is used by EC 1.14.13.106, epi-isozizaene 5-monooxygenase, to produce the sesquiterpene antibiotic albaflavenone [2].
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2E,6E)-farnesyl diphosphate
(+)-epi-isozizaene + diphosphate
show the reaction diagram
(3R)-(Z)-nerolidyl diphosphate
(+)-epi-isozizaene + diphosphate
show the reaction diagram
>93:7 preference for (3R)-nerolidyl diphosphate over (3S)-nerolidyl diphosphate
-
-
?
(2E,6E)-farnesyl diphosphate
(+)-epi-isozizaene + diphosphate
show the reaction diagram
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(2E,6E)-farnesyl diphosphate
(+)-epi-isozizaene + diphosphate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
complete inhibition
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000147 - 0.00145
(2E,6E)-farnesyl diphosphate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00024 - 0.049
(2E,6E)-farnesyl diphosphate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.23 - 63
(2E,6E)-farnesyl diphosphate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
18
recombinant wild-type enzyme, pH 7.5, 30°C
4
recombinant mutants F96S and F96N, pH 7.5, 30°C
4.2
recombinant mutant W230H, pH 7.5, 30°C
5
recombinant mutants F95N and Y69A, pH 7.5, 30°C
5.5
recombinant mutant F95C, pH 7.5, 30°C
5.8
recombinant mutant F96H, pH 7.5, 30°C
5.9
recombinant mutant F96T, pH 7.5, 30°C
5.92
recombinant mutant F95Q, pH 7.5, 30°C
6
recombinant mutant W230Y, pH 7.5, 30°C
7
recombinant mutant F96M, pH 7.5, 30°C
7.6
recombinant mutant Y69F, pH 7.5, 30°C
8
recombinant mutants F96Q and F95Y, pH 7.5, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
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
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
sitting drop vapor diffusion method
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D100N
below 5% of wild-type activity
D99E
below 5% of wild-type activity
E248D
below 5% of wild-type activity
F198A
major products: Z-gamma-bisabolene (24%), sesqusabine (20%)
F96A
major product: beta-farnesene (70%)
N240D
below 5% of wild-type activity
S244A
below 5% of wild-type activity
W203F
major product: Z-gamma-bisabolene (47%)
F95C
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
F95N
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
F95Q
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
F95Y
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
F96H
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
F96M
site-directed mutagenesis, the substitution converts the epi-isozizaene synthase into a high-fidelity sesquisabinene synthase, the mutant generates 91% sesquisabinene A
F96N
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
F96Q
site-directed mutagenesis, the substitution converts the epi-isozizaene synthase into a high-fidelity sesquisabinene synthase, the mutant generates 97% sesquisabinene A
F96S
site-directed mutagenesis, the substitution converts the epi-isozizaene synthase into a high-fidelity sesquisabinene synthase, the mutant generates 78% sesquisabinene A
F96T
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
W203H
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
W203Y
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
Y69A
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
Y69F
site-directed mutagenesis, the mutant shows reduced activity with farnesyl diphosphate compared to the wild-type
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
immobilized metal ion affinity chromatography (Co2+)
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
His-tagged version expressed in Escherichia coli BL21(DE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Lin, X.; Hopson, R.; Cane, D.E.
Genome mining in Streptomyces coelicolor: molecular cloning and characterization of a new sesquiterpene synthase
J. Am. Chem. Soc.
128
6022-6023
2006
Streptomyces coelicolor (Q9K499), Streptomyces coelicolor A3(2) (Q9K499)
Manually annotated by BRENDA team
Aaron, J.A.; Lin, X.; Cane, D.E.; Christianson, D.W.
Structure of epi-isozizaene synthase from Streptomyces coelicolor A3(2), a platform for new terpenoid cyclization templates
Biochemistry
49
1787-1797
2010
Streptomyces coelicolor (Q9K499), Streptomyces coelicolor A3(2) (Q9K499), Streptomyces coelicolor A3(2)
Manually annotated by BRENDA team
Lin, X.; Cane, D.E.
Biosynthesis of the sesquiterpene antibiotic albaflavenone in Streptomyces coelicolor. Mechanism and stereochemistry of the enzymatic formation of epi-isozizaene
J. Am. Chem. Soc.
131
6332-6333
2009
Streptomyces coelicolor (Q9K499), Streptomyces coelicolor, Streptomyces coelicolor A3(2) (Q9K499)
Manually annotated by BRENDA team
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
Biochemistry
56
5798-5811
2017
Streptomyces coelicolor (Q9K498), Streptomyces coelicolor A3(2) (Q9K498)
Manually annotated by BRENDA team
Pemberton, R.P.; Ho, K.C.; Tantillo, D.J.
Modulation of inherent dynamical tendencies of the bisabolyl cation via preorganization in epi-isozizaene synthase
Chem. Sci.
6
2347-2353
2015
Streptomyces coelicolor
Manually annotated by BRENDA team