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geranylgeranyl diphosphate + H2O = cyclooctat-9-en-7-ol + diphosphate
geranylgeranyl diphosphate + H2O = cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O = cyclooctat-9-en-7-ol + diphosphate
cyclization reaction mechanism analysis using three regiospecifically deuterated samples of geranylgeranyl diphosphate, the reaction proceeds via several cationic intermediates, detailed overview. Enzyme CotB2 catalyzes an unusually complex regiospecific and stereospecific cyclization that involves a unique carbon-carbon bond rearrangement and multiple hydride shifts, which all take place at a single active site
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
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
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geranylgeranyl diphosphate + H2O = cyclooctat-9-en-7-ol + diphosphate
cyclization reaction mechanism analysis using three regiospecifically deuterated samples of geranylgeranyl diphosphate, the reaction proceeds via several cationic intermediates, detailed overview. Enzyme CotB2 catalyzes an unusually complex regiospecific and stereospecific cyclization that involves a unique carbon-carbon bond rearrangement and multiple hydride shifts, which all take place at a single active site
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
additional information
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
the enzyme is involved in biosynthesis of cyclooctatin
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
the enzyme is involved in biosynthesis of cyclooctatin
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
the enzyme is involved in biosynthesis of cyclooctatin
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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additional information
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CotB2 has the unique ability to synthesize the characteristic 5-8-5 fused-ring system of cyclooctat-9-en-7-ol having six chiral centers by cyclization of the universal diterpene precursor, the achiral C20 allylic diphosphate GGDP
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additional information
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CotB2-catalyzed cyclization of geranylgeranyl diphosphate to the diterpene cyclooctat-9-en-7-ol
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additional information
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CotB2-catalyzed cyclization of geranylgeranyl diphosphate to the diterpene cyclooctat-9-en-7-ol
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additional information
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CotB2 has the unique ability to synthesize the characteristic 5-8-5 fused-ring system of cyclooctat-9-en-7-ol having six chiral centers by cyclization of the universal diterpene precursor, the achiral C20 allylic diphosphate GGDP
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
the enzyme is involved in biosynthesis of cyclooctatin
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
the enzyme is involved in biosynthesis of cyclooctatin
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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geranylgeranyl diphosphate + H2O
cyclooctat-9-en-7-ol + diphosphate
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evolution
the enzyme belongs to the class I terpene cyclases
evolution
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the enzyme belongs to the class I terpene cyclases
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metabolism
the enzyme is involved in biosynthesis of cyclooctatin
metabolism
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the enzyme is involved in biosynthesis of cyclooctatin
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metabolism
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the enzyme is involved in biosynthesis of cyclooctatin
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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
physiological function
the enzyme catalyzes the formation of cyclooctat-9-en-7-ol, which is a precursor of cyclooctatin, a diterpene with a unique tricyclic diterpene skeleton characterized by a 5-8-5 fused-ring system, which is a potent inhibitor of lysophospholipase, an enzyme that catalyzes the hydrolysis of the fatty acid ester bonds of lysophospholipids
physiological function
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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
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physiological function
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the enzyme catalyzes the formation of cyclooctat-9-en-7-ol, which is a precursor of cyclooctatin, a diterpene with a unique tricyclic diterpene skeleton characterized by a 5-8-5 fused-ring system, which is a potent inhibitor of lysophospholipase, an enzyme that catalyzes the hydrolysis of the fatty acid ester bonds of lysophospholipids
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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
additional information
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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
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F107G
the mutant enzyme produces cembrene A as single cyclization product
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
F149H
the mutant enzyme produces a single cyclization product
F149V
the mutant enzyme produces a single cyclization product
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
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
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
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
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
F107G
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the mutant enzyme produces cembrene A as single cyclization product
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F107L
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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
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F149V
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the mutant enzyme produces a single cyclization product
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N103A
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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
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W186F
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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
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F107A
mutation results in the formation of a monocyclic cembrene, which structurally bears no resemblance to the parent compound cyclooctat-9-en-7-ol
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
F107Y
site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product cyclooctat-1,7-diene
F107Y
the mutant enzyme produces a product mixture
F149L
cyclisation of geranylgeranyl diphosphate by the the mutant enzyme results in the formation of the non-natural fusicoccane macrocycle cyclooctat-7-en-3-ol
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
W288G
site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product 3,7,18-dolabellatriene
W288G
site-directed mutagenesis, the mutant produces (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene instead of the native product cyclooctat-9-en-7-ol. In vivo CotB2 W288G reconstitution in an Escherichia coli based terpene production system, allows efficient production of this olefinic macrocycle
F107A
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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
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F107A
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mutation results in the formation of a monocyclic cembrene, which structurally bears no resemblance to the parent compound cyclooctat-9-en-7-ol
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F107Y
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site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product cyclooctat-1,7-diene
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F107Y
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the mutant enzyme produces a product mixture
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F149L
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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
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F149L
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cyclisation of geranylgeranyl diphosphate by the the mutant enzyme results in the formation of the non-natural fusicoccane macrocycle cyclooctat-7-en-3-ol
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W288G
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site-directed mutagenesis, the mutant follows a different reaction mechanism compared to wild-type and produces the different product 3,7,18-dolabellatriene
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W288G
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site-directed mutagenesis, the mutant produces (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene instead of the native product cyclooctat-9-en-7-ol. In vivo CotB2 W288G reconstitution in an Escherichia coli based terpene production system, allows efficient production of this olefinic macrocycle
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additional information
epoxidation of product (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene from mutant CotB2 W288G by acetic peracid, which is formed in situ by a lipase catalyzed reaction of acetic acid with H2O2, provides efficient access to two monooxidized dolabellanes and to a di-epoxidated dolabellane species. These compounds might act as synthons en-route to other dolabellanes with diversified bioactivities. Almost quantitative 3,7,18-dolabellatriene conversion into the non-natural compound (1R,3E,7E,11S,12S,18R)-dolabella-3,7-diene-20-ol by hydroboration-oxidation with an enantiomeric excess of 94%. Molecular docking of (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene in the P450BM3_F87A and P450BM3_F87A/A328L active site of hydroxylase P450BM3 derived from Bacillus megaterium
additional information
mutational analysis of the atypical aspartate-rich motif of CotB2. Proposed cyclization mechanism for CotB2 and its mutants, overview
additional information
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mutational analysis of the atypical aspartate-rich motif of CotB2. Proposed cyclization mechanism for CotB2 and its mutants, overview
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additional information
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epoxidation of product (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene from mutant CotB2 W288G by acetic peracid, which is formed in situ by a lipase catalyzed reaction of acetic acid with H2O2, provides efficient access to two monooxidized dolabellanes and to a di-epoxidated dolabellane species. These compounds might act as synthons en-route to other dolabellanes with diversified bioactivities. Almost quantitative 3,7,18-dolabellatriene conversion into the non-natural compound (1R,3E,7E,11S,12S,18R)-dolabella-3,7-diene-20-ol by hydroboration-oxidation with an enantiomeric excess of 94%. Molecular docking of (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene in the P450BM3_F87A and P450BM3_F87A/A328L active site of hydroxylase P450BM3 derived from Bacillus megaterium
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Zhang, X.;, Shang, G.; Gu, L.; Shen Y.
Crystallization and preliminary X-ray diffraction analysis of the diterpene cyclooctatin synthase (CYC) from Streptomyces sp. LZ35
Acta Crystallogr. Sect. F
70
366-369
2014
Streptomyces sp., Streptomyces sp. LZ35
brenda
Kim, S.Y.; Zhao, P.; Igarashi, M.; Sawa, R.; Tomita, T.; Nishiyama, M.; Kuzuyama, T.
Cloning and heterologous expression of the cyclooctatin biosynthetic gene cluster afford a diterpene cyclase and two p450 hydroxylases
Chem. Biol.
16
736-743
2009
Streptomyces melanosporofaciens, Streptomyces melanosporofaciens MI614-43F2
brenda
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
ACS Chem. Biol.
12
1621-1628
2017
Streptomyces melanosporofaciens (C9K1X5), Streptomyces melanosporofaciens MI614-43F2 (C9K1X5)
brenda
Meguro, A.; Motoyoshi, Y.; Teramoto, K.; Ueda, S.; Totsuka, Y.; Ando, Y.; Tomita, T.; Kim, S.Y.; Kimura, T.; Igarashi, M.; Sawa, R.; Shinada, T.; Nishiyama, M.; Kuzuyama, T.
An unusual terpene cyclization mechanism involving a carbon-carbon bond rearrangement
Angew. Chem. Int. Ed. Engl.
54
4353-4356
2015
Streptomyces melanosporofaciens (C9K1X5), Streptomyces melanosporofaciens MI614-43F2 (C9K1X5)
brenda
Kim, S.; Zhao, P.; Igarashi, M.; Sawa, R.; Tomita, T.; Nishiyama, M.; Kuzuyama, T.
Cloning and heterologous expression of the cyclooctatin biosynthetic gene cluster affordxa0a diterpene cyclase and two P450 hydroxylases
Chem. Biol.
16
736-743
2009
Streptomyces melanosporofaciens (C9K1X5), Streptomyces melanosporofaciens M1614-43f2 (C9K1X5)
brenda
Grner, C.; Huslein, I.; Schrepfer, P.; Eisenreich, W.; Brck, T.
Targeted engineering of cyclooctat-9-en-7-ol synthase A stereospecific access to two new non-natural fusicoccane-type diterpenes
ChemCatChem
5
3289-3298
2013
Streptomyces melanosporofaciens (C9K1X5), Streptomyces melanosporofaciens MI614-43F2 (C9K1X5)
-
brenda
Goerner, C.; Hirte, M.; Huber, S.; Schrepfer, P.; Brueck, T.
Stereoselective chemo-enzymatic oxidation routes for (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene
Front. Microbiol.
6
1115
2015
Streptomyces melanosporofaciens (C9K1X5), Streptomyces melanosporofaciens MI614-43F2 (C9K1X5)
brenda