Literature summary for 4.2.3.146 extracted from

Tomita, T.; Kim, S.Y.; Teramoto, K.; Meguro, A.; Ozaki, T.; Yoshida, A.; Motoyoshi, Y.; Mori, N.; Ishigami, K.; Watanabe, H.; Nishiyama, M.; Kuzuyama, T.
Structural insights into the CotB2-catalyzed cyclization of geranylgeranyl diphosphate to the diterpene cyclooctat-9-en-7-ol (2017), ACS Chem. Biol., 12, 1621-1628 .

Search for more literature for 4.2.3.146

Cloned(Commentary)

Cloned (Comment)	Organism
gene cotB2, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), selenomethionine-substituted CotB2 was expressed in Escherichia coli strain B834(DE3)	Streptomyces melanosporofaciens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant native and SeMet-labeled enzyme CotB2 with bound substrate analogue geranylgeranyl thiodiphosphate, hanging drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein, in 20 mM Tris-HCl, pH 8.0, 5 mM MgSO4, with or without 1 mM substrate, with 0.001 ml of reservoir solution, and equilibration against 0.5 ml of reservoir solution, 20°C, the reservoir solution contains 0.1 M HEPES-NaOH, pH 7.0-8.5, and 1.4-1.5 M ammonium formate for the apoenzyme, 0.1 M Tris-HCl, pH 8.0, and 2.4 M ammonium formate for the apo-SeMet-enzyme, and 0.1 M bicine-NaOH, pH 9.0, and 20% PEG 6000 for the substrate-bound enzyme, X-ray diffraction structure determination and analysis. Molecular replacement. Only a single Mg2+ ion is present in the observed structure, coordinated by the Asn220, Ser224, and Glu228 side chains belonging to the canonical NSE/DTE motif	Streptomyces melanosporofaciens

Crystallization (Comment)

Organism

purified recombinant native and SeMet-labeled enzyme CotB2 with bound substrate analogue geranylgeranyl thiodiphosphate, hanging drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein, in 20 mM Tris-HCl, pH 8.0, 5 mM MgSO4, with or without 1 mM substrate, with 0.001 ml of reservoir solution, and equilibration against 0.5 ml of reservoir solution, 20°C, the reservoir solution contains 0.1 M HEPES-NaOH, pH 7.0-8.5, and 1.4-1.5 M ammonium formate for the apoenzyme, 0.1 M Tris-HCl, pH 8.0, and 2.4 M ammonium formate for the apo-SeMet-enzyme, and 0.1 M bicine-NaOH, pH 9.0, and 20% PEG 6000 for the substrate-bound enzyme, X-ray diffraction structure determination and analysis. Molecular replacement. Only a single Mg2+ ion is present in the observed structure, coordinated by the Asn220, Ser224, and Glu228 side chains belonging to the canonical NSE/DTE motif

Streptomyces melanosporofaciens

Protein Variants

Protein Variants	Comment	Organism
F107A	site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product cembrane A in addition to cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens
F107L	site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different products 3,7-dolabellatriene-9-ol and cyclooctat-6-en-8-ol in addition to cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens
F107Y	site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product cyclooctat-1,7-diene	Streptomyces melanosporofaciens
F149L	site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product cyclooctat-7-en-3-ol, no formation of cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens
F185A	site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different products 3,7-dolabellatriene-9-ol and cyclooctat-6-en-8-ol in addition to cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens
additional information	mutational analysis of the atypical aspartate-rich motif of CotB2. Proposed cyclization mechanism for CotB2 and its mutants, overview	Streptomyces melanosporofaciens
N103A	site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product 3,7,12-dolabellatriene instead of cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens
W186F	site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different products 3,7-dolabellatriene-9-ol and cyclooctat-6-en-8-ol in addition to cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens
W186H	site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product 3,7,18-dolabellatriene instead of cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens
W186L	site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different products cembrane A and 3,7,18-dolabellatriene and only low amounts of cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens
W288G	site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product 3,7,18-dolabellatriene	Streptomyces melanosporofaciens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required, in the substrate-bound form, a Mg2+ ion is coordinated in an octahedral manner by the Asn220, Ser224, and Glu228 side chains of the NSE motif, as well as by a water molecule and the diphosphate group of GGSPP. Compared to the apoform, the side chain of Asn220 is rotated and oriented to allow Mg2+ binding	Streptomyces melanosporofaciens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
geranylgeranyl diphosphate + H2O	Streptomyces melanosporofaciens	-	cyclooctat-9-en-7-ol + diphosphate	-	?
geranylgeranyl diphosphate + H2O	Streptomyces melanosporofaciens MI614-43F2	-	cyclooctat-9-en-7-ol + diphosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Streptomyces melanosporofaciens	C9K1X5	-	-
Streptomyces melanosporofaciens MI614-43F2	C9K1X5	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, ultrafiltration, and gel filtration to over 95% purity	Streptomyces melanosporofaciens

Reaction

Reaction	Comment	Organism	Reaction ID
geranylgeranyl diphosphate + H2O = cyclooctat-9-en-7-ol + diphosphate	proposed cyclization mechanism for CotB2. Bound to the enzyme, geranylgeranyl dithiophosphate (GGSPP) is folded into a unique S-shaped conformation in the active site pocket. GGSPP cannot undergo ionization and generate a reactive carbocation intermediate due to the C-S bond that links the thiodiphosphate moiety and the C20 chain. Therefore, the observed conformation of bound GGSPP is thought to represent the preionization state of the natural CotB2 substrate geranylgeranyl diphosphate	Streptomyces melanosporofaciens	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
geranylgeranyl diphosphate + H2O	-	Streptomyces melanosporofaciens	cyclooctat-9-en-7-ol + diphosphate	-	?
geranylgeranyl diphosphate + H2O	when bound to the enzyme, geranylgeranyl dithiophosphate (GGSPP) is folded in the active site pocket into a unique S-shaped conformation. A Mg2+ ion is coordinated in an octahedral manner by the Asn220, Ser224, and Glu228 side chains of the NSE motif, as well as by a water molecule and the diphosphate group of GGSPP. Compared to the apoform, the side chain of Asn220 is rotated and oriented to allow Mg2+ binding. The diphosphate group of GGSPP is also recognized by Arg177, Arg227, and the basic motif residues Arg294 and Tyr295. Active site docking model for the complex of CotB2 and reaction product (2R,3R,6R,7S,11R,14R)-cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens	cyclooctat-9-en-7-ol + diphosphate	-	?
geranylgeranyl diphosphate + H2O	-	Streptomyces melanosporofaciens MI614-43F2	cyclooctat-9-en-7-ol + diphosphate	-	?
geranylgeranyl diphosphate + H2O	when bound to the enzyme, geranylgeranyl dithiophosphate (GGSPP) is folded in the active site pocket into a unique S-shaped conformation. A Mg2+ ion is coordinated in an octahedral manner by the Asn220, Ser224, and Glu228 side chains of the NSE motif, as well as by a water molecule and the diphosphate group of GGSPP. Compared to the apoform, the side chain of Asn220 is rotated and oriented to allow Mg2+ binding. The diphosphate group of GGSPP is also recognized by Arg177, Arg227, and the basic motif residues Arg294 and Tyr295. Active site docking model for the complex of CotB2 and reaction product (2R,3R,6R,7S,11R,14R)-cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens MI614-43F2	cyclooctat-9-en-7-ol + diphosphate	-	?
additional information	CotB2-catalyzed cyclization of geranylgeranyl diphosphate to the diterpene cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens	?	-	?
additional information	CotB2-catalyzed cyclization of geranylgeranyl diphosphate to the diterpene cyclooctat-9-en-7-ol	Streptomyces melanosporofaciens MI614-43F2	?	-	?

Synonyms

Synonyms	Comment	Organism
CotB2	-	Streptomyces melanosporofaciens

Temperature Optimum [°C]

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

pH Optimum

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

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the class I terpene cyclases	Streptomyces melanosporofaciens
additional information	class I terpene cyclases contain two conserved motifs, DDXXD and (N,D)XX(S,T)XXX(E,D), in which residues cooperate to coordinate to the three bound Mg2+ ions that themselves bind and orient the substrate through its diphosphate moiety while facilitating the ionization of the C-O bond of the allylic diphosphate substrate, thereby initiating the cyclization cascade by the generation of a highly reactive carbocation intermediate. CotB2 has an unusual aspartate-rich motif (110DDMD), in which the interval between the second and third aspartate residues is only one residue, whereas typical terpene cyclases have two (DDXXD) or three (DDXXXD) intervening residues. The NSE/DTE motif (220NDFYSYDRE), which is involved in binding Mg2+ ions in class I terpene cyclases, is located opposite the aspartate-rich motif at the upper rim of the CotB2 active site. Stucture-function relationship, overview	Streptomyces melanosporofaciens
physiological function	the diterpene cyclase CotB2 catalyzes the cyclization of acyclic geranylgeranyl diphosphate (GGPP) to produce tricyclic (2R,3R,6R,7S,11R,14R)-cyclooctat-9-en-7-ol, which is characterized by a 5-8-5-fused ring skeleton and constitutes the carbon framework of a potent lisophospholipase inhibitor, cyclooctatin. Proposal of a cyclization cascade involving a unique carbon-carbon bond rearrangement combined with multiple hydride shifts, all occurring at a single active site. The enzyme exhibits effective control of ring formation and stereochemistry during CotB2 catalysis	Streptomyces melanosporofaciens