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Literature summary extracted from

  • Piechulla, B.; Bartelt, R.; Brosemann, A.; Effmert, U.; Bouwmeester, H.; Hippauf, F.; Brandt, W.
    The alpha-terpineol to 1,8-cineole cyclization reaction of tobacco terpene synthases (2016), Plant Physiol., 172, 2120-2131 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.3.108 DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant overexpression of His-tagged wild-type and mutant enzymes in Escherichia coli strain HMS 174(DE3) Nicotiana suaveolens
4.2.3.108 DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant overexpression of His-tagged wild-type enzyme in Escherichia coli strain HMS174(DE3) Nicotiana forgetiana
4.2.3.111 gene TER, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant overexpression of His-tagged wild-type enzyme in Escherichia coli strain HMS 174(DE3) Nicotiana langsdorffii

Protein Variants

EC Number Protein Variants Comment Organism
4.2.3.108 F266C site-directed mutagenesis, the mutation only slightly alters the product spectrum of the mutant enzyme compared to wild-type enzyme Nicotiana suaveolens
4.2.3.108 F266S site-directed mutagenesis, the mutation shifts the the product spectrum significantly toward alpha-terpineol compared to wild-type enzyme Nicotiana suaveolens
4.2.3.108 F266T site-directed mutagenesis, the mutation only slightly alters the product spectrum of the mutant enzyme compared to wild-type enzyme Nicotiana suaveolens
4.2.3.108 F266V site-directed mutagenesis, the mutation only slightly alters the product spectrum of the mutant enzyme compared to wild-type enzyme Nicotiana suaveolens
4.2.3.108 F266Y site-directed mutagenesis, the mutation only slightly alters the product spectrum of the mutant enzyme compared to wild-type enzyme Nicotiana suaveolens
4.2.3.108 additional information although the distributions of the five detectable products sabinene, beta-myrcene, limonene, 1,8-cineole, and alpha-terpineol change slightly in all these mutants compared to wild-type, none of them shifts the product spectrum significantly toward alpha-terpineol, with exception of mutant F266S, overview Nicotiana suaveolens
4.2.3.108 N419A site-directed mutagenesis, the mutation results in a drastic drop of enzyme activity, and except for traces of alpha-terpineol, no cyclic products are detected Nicotiana forgetiana
4.2.3.108 T278A site-directed mutagenesis, the product composition of the Thr mutant is altered compared to the wild-type enzyme, the most striking change is the decreased amount of 1,8-cineole. Thus, this mutation converts the wild-type cineole synthase into an alpha-terpineol synthase. Assuming that alpha-terpineol is a distinct precursor in the biosynthesis of 1,8-cineole, a decrease of cineole within the product profile indicates a disturbed reaction mechanism of the cyclization of alpha-terpineol toward 1,8-cineole Nicotiana forgetiana
4.2.3.108 T279A site-directed mutagenesis, the mutation of this residue does not change the product composition but leads to an overall increase of activity in Nicotiana forgetiana Nicotiana forgetiana
4.2.3.108 W253A site-directed mutagenesis, the mutant reveals a strongly decreased amount of cyclic monoterpenes Nicotiana forgetiana
4.2.3.108 W253M site-directed mutagenesis, an exchange to Met does not seem to provide a comparable stabilization Nicotiana forgetiana
4.2.3.108 Y496F site-directed mutagenesis, mutation of the catalytic Tyr causes a drastic decrease of cyclic products Nicotiana forgetiana
4.2.3.111 A147R/A148N site-directed mutagenesis, the mutation leads to an altered monoterpenes profile compared to wild-type Nicotiana langsdorffii
4.2.3.111 A277T/A147R/A148N site-directed mutagenesis, the mutation leads to an altered monoterpenes profile compared to wild-type Nicotiana langsdorffii
4.2.3.111 S264F site-directed mutagenesis, the mutation leads to an altered monoterpenes profile compared to wild-type Nicotiana langsdorffii

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.2.3.108 0.00004
-
 geranyl diphosphate recombinant mutant F266T, pH and temperature not specified in the publication Nicotiana suaveolens
4.2.3.108 0.00004
-
 geranyl diphosphate recombinant mutant F266V, pH and temperature not specified in the publication Nicotiana suaveolens
4.2.3.108 0.00012
-
 geranyl diphosphate recombinant mutant F266S, pH and temperature not specified in the publication Nicotiana suaveolens
4.2.3.108 0.00019
-
 geranyl diphosphate recombinant wild-type enzyme, pH and temperature not specified in the publication Nicotiana suaveolens

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
4.2.3.108 chloroplast
-
 Nicotiana forgetiana 9507
-
4.2.3.111 chloroplast
-
 Nicotiana langsdorffii 9507
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.3.108 Mg2+ required Nicotiana suaveolens
4.2.3.108 Mg2+ required Nicotiana forgetiana
4.2.3.111 Mg2+ required Nicotiana langsdorffii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.3.108 geranyl diphosphate + H2O Nicotiana suaveolens
-
 1,8-cineole + diphosphate
-
 ?
4.2.3.108 geranyl diphosphate + H2O Nicotiana forgetiana
-
 1,8-cineole + diphosphate
-
 ?
4.2.3.111 geranyl diphosphate + H2O Nicotiana langsdorffii
-
 (-)-alpha-terpineol + diphosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
4.2.3.108 Nicotiana forgetiana I7CTV3
-
-
4.2.3.108 Nicotiana suaveolens A5Y5L5
-
-
4.2.3.111 Nicotiana langsdorffii H2ELN1
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.3.108 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain HMS 174(DE3) by nickel affinity chromaatography Nicotiana suaveolens
4.2.3.108 recombinant His-tagged wild-type enzyme from Escherichia coli strain HMS 174(DE3) by nickel affinity chromatography Nicotiana forgetiana
4.2.3.111 recombinant His-tagged wild-type enzyme from Escherichia coli strain HMS 174(DE3) by nickel affinity chromatography Nicotiana langsdorffii

Reaction

EC Number Reaction Comment Organism Reaction ID
4.2.3.108 geranyl diphosphate + H2O = 1,8-cineole + diphosphate proposed mechanism for the formation of 1,8-cineole, the reaction can proceed via (R)-alpha-terpinyl cation and (R)-alpha-terpineol, as well as via (S)-alpha-terpinyl cation and (S)-alpha-terpineol, the catalytic dyad is formed by H502 and E249. Depending on the stereochemistry of the intermediate, the protonation of the double bond of the alpha-terpinyl cation is provided by a proton relay via the hydroxyl groups of either Tyr496 or Thr278. The substrate GPP is ionized by diphosphate elimination, resulting in the geranyl cation. Subsequently, this cation is converted into the linalyl cation and alpha-terpinyl cation. The intermediate alpha-terpinyl cation is the precursor for all cyclic monoterpenes. 1,8-Cineol is possibly directly formed from geranyl diphosphate, and a cyclization reaction resulting in 1,8-cineole uses alpha-terpineol as a precursor. alpha-Terpineol is formed after water capture of the alpha-terpinyl cation Nicotiana forgetiana
a
4.2.3.108 geranyl diphosphate + H2O = 1,8-cineole + diphosphate the substrate GPP is ionized by diphosphate elimination, resulting in the geranyl cation. Subsequently, this cation is converted into the linalyl cation and alpha-terpinyl cation. The intermediate alpha-terpinyl cation is the precursor for all cyclic monoterpenes. 1,8-Cineol is possibly directly formed from geranyl diphosphate, and a cyclization reaction resulting in 1,8-cineole uses alpha-terpineol as a precursor. alpha-Terpineol is formed after water capture of the alpha-terpinyl cation. Proposed mechanism for the formation of 1,8-cineole Nicotiana suaveolens
a
4.2.3.111 geranyl diphosphate + H2O = (-)-alpha-terpineol + diphosphate the substrate geranyl diphosphate is ionized by diphosphate elimination, resulting in the geranyl cation. Subsequently, this cation is converted into the linalyl cation and alpha-terpinyl cation. The intermediate alpha-terpinyl cation is the precursor for all cyclic monoterpenes. alpha-Terpineol is formed after water capture of the alpha-terpinyl cation. A cyclization reaction resulting in 1,8-cineole uses alpha-terpineol as a precursor Nicotiana langsdorffii
a

Source Tissue

EC Number Source Tissue Comment Organism Textmining
4.2.3.108 leaf
-
 Nicotiana suaveolens
-
4.2.3.108 leaf
-
 Nicotiana forgetiana
-
4.2.3.111 leaf
-
 Nicotiana langsdorffii
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.3.108 geranyl diphosphate + H2O
-
 Nicotiana suaveolens 1,8-cineole + diphosphate
-
 ?
4.2.3.108 geranyl diphosphate + H2O
-
 Nicotiana forgetiana 1,8-cineole + diphosphate
-
 ?
4.2.3.108 geranyl diphosphate + H2O the enzyme releases alpha-terpineol as the main compound as a terpineol synthase (TER). The S to R ratio is 6.6:1 and 6.5:1 in two experimental passages Nicotiana suaveolens 1,8-cineole + diphosphate
-
 ?
4.2.3.108 geranyl diphosphate + H2O the enzyme releases alpha-terpineol as the main compound as a terpineol synthase (TER). The S to R ratio is 7.9:1 and 7.7:1 in two experimental passages Nicotiana forgetiana 1,8-cineole + diphosphate
-
 ?
4.2.3.108 additional information the formation of alpha-terpineol starts by a nucleophilic attack of water. During this attack, the alpha-terpinyl cation is stabilized by Pi-stacking with a tryptophan side chain (Tryp253). The hypothesized catalytic mechanism of alpha-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (His502 and Glu249), acting as a base, and a threonine (Thr278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (2S)-2-alpha-terpineol, which is the favored enantiomer product of the recombinant enzymes. Product analysis and quantification by GC-MS. Binding structure of the reactive intermediate alpha-terpinyl cation in the active site of cineole synthase involving residues Trp253, His502, and Thr278, overview. The hydroxyl group of Tyr496 is necessary to control the orientation of Asn419. This Asn itself is proposed to be involved in binding and fixation of the diphosphate moiety of the substrate. Major role of Thr278 in the formation of cineole by fixing the intermediate alpha-terpineol and supporting the autoprotonation of its double bond. Residue Phe266 is relevant for the product outcome of the 1,8-cineole synthase Nicotiana forgetiana ?
-
 ?
4.2.3.108 additional information the formation of alpha-terpineol strats by a nucleophilic attack of water. During this attack, the alpha-terpinyl cation is stabilized by Pi-stacking with a tryptophan side chain (Tryp253). The hypothesized catalytic mechanism of alpha-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (His502 and Glu249), acting as a base, and a threonine (Thr278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (2S)-2-alpha-terpineol, which is the favored enantiomer product of the recombinant enzymes. Product analysis and quantification by GC-MS Nicotiana suaveolens ?
-
 ?
4.2.3.111 geranyl diphosphate + H2O
-
 Nicotiana langsdorffii (-)-alpha-terpineol + diphosphate
-
 ?
4.2.3.111 geranyl diphosphate + H2O the enzyme releases alpha-terpineol as the main compound as a terpineol synthase (TER). The S to R ratio is 11.2:1 and 10.8:1 in two experimental passages Nicotiana langsdorffii (-)-alpha-terpineol + diphosphate
-
 ?
4.2.3.111 additional information the formation of alpha-terpineol starts by a nucleophilic attack of water. During this attack, the alpha-terpinyl cation is stabilized by Pi-stacking with a tryptophan side chain (Tryp253). The hypothesized catalytic mechanism of alpha-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (His502 and Glu249), acting as a base, and a threonine (Thr278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (2S)-2-alpha-terpineol, which is the favored enantiomer product of the recombinant enzymes. Product analysis and quantification by GC-MS Nicotiana langsdorffii ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
4.2.3.108 CIN
-
 Nicotiana forgetiana
4.2.3.108 TER
-
 Nicotiana suaveolens
4.2.3.111 TER
-
 Nicotiana langsdorffii

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.3.108 0.000058
-
 geranyl diphosphate recombinant mutant F266T, pH and temperature not specified in the publication Nicotiana suaveolens
4.2.3.108 0.00008
-
 geranyl diphosphate recombinant mutant F266V, pH and temperature not specified in the publication Nicotiana suaveolens
4.2.3.108 0.00029
-
 geranyl diphosphate recombinant mutant F266S, pH and temperature not specified in the publication Nicotiana suaveolens
4.2.3.108 0.00041
-
 geranyl diphosphate recombinant wild-type enzyme, pH and temperature not specified in the publication Nicotiana suaveolens

General Information

EC Number General Information Comment Organism
4.2.3.108 evolution the amounts and ratios of alpha-terpineol enantiomers is species-specific for cineole synthases and terpineol synthases of different species, overview. For Nicotinana forgetiana, the S:R ratio ((S)-(-)-alpha-terpineol to (R)-(+)-alpha-terpineol) is 7.7-7.9:1 Nicotiana forgetiana
4.2.3.108 metabolism cyclization reactions of monoterpene synthases, overview. Substrate GPP is ionized by diphosphate elimination, resulting in the geranyl cation. Subsequently, this cation is converted into the linalyl cation and alpha-terpinyl cation. The synthesis of the acyclic beta-myrcene might proceed via the geranyl cation or via the linalyl cation by deprotonation. The intermediate alpha-terpinyl cation is the precursor for all cyclic monoterpenes. The 2,7-ring closure results in the pinyl cation, which is deprotonated to synthesize beta-pinene and alpha-pinene. Sabinene, with a cyclopropane ring, is released after two carbocation formations and 2,6-ring closure. alpha-Terpineol is formed after water capture of the alpha-terpinyl cation. Broken lines indicate possible reactions leading to 1,8-cineole. A cyclization reaction resulting in 1,8-cineole uses alpha-terpineol as a precursor. Enzyme structure homology modelling Nicotiana suaveolens
4.2.3.108 metabolism cyclization reactions of monoterpene synthases, overview. Substrate GPP is ionized by diphosphate elimination, resulting in the geranyl cation. Subsequently, this cation is converted into the linalyl cation and alpha-terpinyl cation. The synthesis of the acyclic beta-myrcene might proceed via the geranyl cation or via the linalyl cation by deprotonation. The intermediate alpha-terpinyl cation is the precursor for all cyclic monoterpenes. The 2,7-ring closure results in the pinyl cation, which is deprotonated to synthesize beta-pinene and alpha-pinene. Sabinene, with a cyclopropane ring, is released after two carbocation formations and 2,6-ring closure. alpha-Terpineol is formed after water capture of the alpha-terpinyl cation. Broken lines indicate possible reactions leading to 1,8-cineole. A cyclization reaction resulting in 1,8-cineole uses alpha-terpineol as a precursor. Enzyme structure homology modelling Nicotiana forgetiana
4.2.3.108 additional information the amino acids at positions 147, 148, and 266 determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase Nicotiana suaveolens
4.2.3.108 additional information the amino acids at positions 147, 148, and 266 determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase. Homology modeling of 1,8-cineole synthases of Nicotiana forgetiana and Nicotiana suaveolens Nicotiana forgetiana
4.2.3.111 metabolism cyclization reactions of monoterpene synthases, overview. Substrate geranyl diphosphate is ionized by diphosphate elimination, resulting in the geranyl cation. Subsequently, this cation is converted into the linalyl cation and alpha-terpinyl cation. The synthesis of the acyclic beta-myrcene might proceed via the geranyl cation or via the linalyl cation by deprotonation. The intermediate alpha-terpinyl cation is the precursor for all cyclic monoterpenes. The 2,7-ring closure results in the pinyl cation, which is deprotonated to synthesize beta-pinene and alpha-pinene. Sabinene, with a cyclopropane ring, is released after two carbocation formations and 2,6-ring closure. alpha-Terpineol is formed after water capture of the alpha-terpinyl cation. Broken lines indicate possible reactions leading to 1,8-cineole. A cyclization reaction resulting in 1,8-cineole uses alpha-terpineol as a precursor Nicotiana langsdorffii
4.2.3.111 additional information the amino acids at positions 147, 148, and 266 determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase Nicotiana langsdorffii

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
4.2.3.108 1.45
-
 geranyl diphosphate recombinant mutant F266T, pH and temperature not specified in the publication Nicotiana suaveolens
4.2.3.108 2
-
 geranyl diphosphate recombinant mutant F266V, pH and temperature not specified in the publication Nicotiana suaveolens
4.2.3.108 2.157
-
 geranyl diphosphate recombinant wild-type enzyme, pH and temperature not specified in the publication Nicotiana suaveolens
4.2.3.108 2.416
-
 geranyl diphosphate recombinant mutant F266S, pH and temperature not specified in the publication Nicotiana suaveolens