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 |
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 |
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 |
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 |
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 | - |
? |
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 | - |
- |
- |
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 |
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 |
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 | ? | - |
? |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
4.2.3.146 | CotB2 | - |
Streptomyces melanosporofaciens |
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 |
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 |
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 |