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Literature summary 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 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.3.146 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
4.2.3.150 gene cotB2, recombinant expression of His-tagged mutant enzymes in Escherichia coli strain BL21(DE3) Streptomyces melanosporofaciens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
4.2.3.146 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

EC Number Protein Variants Comment Organism
4.2.3.146 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
4.2.3.146 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
4.2.3.146 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
4.2.3.146 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
4.2.3.146 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
4.2.3.146 additional information mutational analysis of the atypical aspartate-rich motif of CotB2. Proposed cyclization mechanism for CotB2 and its mutants, overview Streptomyces melanosporofaciens
4.2.3.146 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
4.2.3.146 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
4.2.3.146 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
4.2.3.146 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
4.2.3.146 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
4.2.3.150 F107A site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product cembrane A instead of cyclooctat-9-en-7-ol Streptomyces melanosporofaciens
4.2.3.150 additional information mutational analysis of the atypical aspartate-rich motif of CotB2 (EC 4.2.3.146). The substrate specificity of the CotB2 mutants can be completely changed. Proposed cyclization mechanism for CotB2 and its mutants, overview Streptomyces melanosporofaciens
4.2.3.150 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

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.3.146 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
4.2.3.150 Mg2+ required Streptomyces melanosporofaciens

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
4.2.3.146 Streptomyces melanosporofaciens C9K1X5
-
-
4.2.3.146 Streptomyces melanosporofaciens MI614-43F2 C9K1X5
-
-
4.2.3.150 Streptomyces melanosporofaciens
-
-
-
4.2.3.150 Streptomyces melanosporofaciens MI614-43F2
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.3.146 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
4.2.3.150 recombinant His-tagged mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Streptomyces melanosporofaciens

Reaction

EC Number Reaction Comment Organism Reaction ID
4.2.3.146 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

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.3.146 geranylgeranyl diphosphate + H2O
-
Streptomyces melanosporofaciens cyclooctat-9-en-7-ol + diphosphate
-
?
4.2.3.146 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
-
?
4.2.3.146 geranylgeranyl diphosphate + H2O
-
Streptomyces melanosporofaciens MI614-43F2 cyclooctat-9-en-7-ol + diphosphate
-
?
4.2.3.146 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
-
?
4.2.3.146 additional information CotB2-catalyzed cyclization of geranylgeranyl diphosphate to the diterpene cyclooctat-9-en-7-ol Streptomyces melanosporofaciens ?
-
?
4.2.3.146 additional information CotB2-catalyzed cyclization of geranylgeranyl diphosphate to the diterpene cyclooctat-9-en-7-ol Streptomyces melanosporofaciens MI614-43F2 ?
-
?
4.2.3.150 geranylgeranyl diphosphate
-
Streptomyces melanosporofaciens cembrene A + diphosphate
-
?
4.2.3.150 geranylgeranyl diphosphate activity of enzyme mutants F107A and W186L, not of wild-type enzyme Streptomyces melanosporofaciens cembrene A + diphosphate
-
?
4.2.3.150 geranylgeranyl diphosphate
-
Streptomyces melanosporofaciens MI614-43F2 cembrene A + diphosphate
-
?
4.2.3.150 geranylgeranyl diphosphate activity of enzyme mutants F107A and W186L, not of wild-type enzyme Streptomyces melanosporofaciens MI614-43F2 cembrene A + diphosphate
-
?
4.2.3.150 additional information the mutants F107A and W186L follow a different reaction mechanism compared to wild-type CotB2, and produce the different product cembrane A Streptomyces melanosporofaciens ?
-
?
4.2.3.150 additional information the mutants F107A and W186L follow a different reaction mechanism compared to wild-type CotB2, and produce the different product cembrane A Streptomyces melanosporofaciens MI614-43F2 ?
-
?

Synonyms

EC Number Synonyms Comment Organism
4.2.3.146 CotB2
-
Streptomyces melanosporofaciens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.3.146 30
-
assay at Streptomyces melanosporofaciens
4.2.3.150 30
-
assay at Streptomyces melanosporofaciens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.3.146 7.5
-
assay at Streptomyces melanosporofaciens
4.2.3.150 7.5
-
assay at Streptomyces melanosporofaciens

General Information

EC Number General Information Comment Organism
4.2.3.146 evolution the enzyme belongs to the class I terpene cyclases Streptomyces melanosporofaciens
4.2.3.146 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
4.2.3.146 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