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Literature summary for 4.2.3.108 extracted from
- The alpha-terpineol to 1,8-cineole cyclization reaction of tobacco terpene synthases (2016), Plant Physiol., 172, 2120-2131 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| 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 |
| 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 |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| F266C | site-directed mutagenesis, the mutation only slightly alters the product spectrum of the mutant enzyme compared to wild-type enzyme | Nicotiana suaveolens |
| F266S | site-directed mutagenesis, the mutation shifts the the product spectrum significantly toward alpha-terpineol compared to wild-type enzyme | Nicotiana suaveolens |
| F266T | site-directed mutagenesis, the mutation only slightly alters the product spectrum of the mutant enzyme compared to wild-type enzyme | Nicotiana suaveolens |
| F266V | site-directed mutagenesis, the mutation only slightly alters the product spectrum of the mutant enzyme compared to wild-type enzyme | Nicotiana suaveolens |
| F266Y | site-directed mutagenesis, the mutation only slightly alters the product spectrum of the mutant enzyme compared to wild-type enzyme | Nicotiana suaveolens |
| 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 |
| 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 |
| 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 |
| 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 |
| W253A | site-directed mutagenesis, the mutant reveals a strongly decreased amount of cyclic monoterpenes | Nicotiana forgetiana |
| W253M | site-directed mutagenesis, an exchange to Met does not seem to provide a comparable stabilization | Nicotiana forgetiana |
| Y496F | site-directed mutagenesis, mutation of the catalytic Tyr causes a drastic decrease of cyclic products | Nicotiana forgetiana |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.00004 | - |
geranyl diphosphate | recombinant mutant F266T, pH and temperature not specified in the publication | Nicotiana suaveolens |  |
| 0.00004 | - |
geranyl diphosphate | recombinant mutant F266V, pH and temperature not specified in the publication | Nicotiana suaveolens | |
| 0.00012 | - |
geranyl diphosphate | recombinant mutant F266S, pH and temperature not specified in the publication | Nicotiana suaveolens | |
| 0.00019 | - |
geranyl diphosphate | recombinant wild-type enzyme, pH and temperature not specified in the publication | Nicotiana suaveolens | |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| chloroplast | - |
Nicotiana forgetiana | 9507 | - |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Nicotiana suaveolens | |
| Mg2+ | required | Nicotiana forgetiana | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| geranyl diphosphate + H2O | Nicotiana suaveolens | - |
1,8-cineole + diphosphate | - |
? | |
| geranyl diphosphate + H2O | Nicotiana forgetiana | - |
1,8-cineole + diphosphate | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Nicotiana forgetiana | I7CTV3 | - |
- |
| Nicotiana suaveolens | A5Y5L5 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain HMS 174(DE3) by nickel affinity chromaatography | Nicotiana suaveolens |
| recombinant His-tagged wild-type enzyme from Escherichia coli strain HMS 174(DE3) by nickel affinity chromatography | Nicotiana forgetiana |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| 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 | |
| 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 |
Source Tissue
| Source Tissue | Comment | Organism | Textmining |
|---|---|---|---|
| leaf | - |
Nicotiana suaveolens | - |
| leaf | - |
Nicotiana forgetiana | - |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| geranyl diphosphate + H2O | - |
Nicotiana suaveolens | 1,8-cineole + diphosphate | - |
? | |
| geranyl diphosphate + H2O | - |
Nicotiana forgetiana | 1,8-cineole + diphosphate | - |
? | |
| 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 | - |
? | |
| 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 | - |
? | |
| 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 | ? | - |
? | |
| 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 | ? | - |
? | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| CIN | - |
Nicotiana forgetiana |
| TER | - |
Nicotiana suaveolens |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.000058 | - |
geranyl diphosphate | recombinant mutant F266T, pH and temperature not specified in the publication | Nicotiana suaveolens | |
| 0.00008 | - |
geranyl diphosphate | recombinant mutant F266V, pH and temperature not specified in the publication | Nicotiana suaveolens | |
| 0.00029 | - |
geranyl diphosphate | recombinant mutant F266S, pH and temperature not specified in the publication | Nicotiana suaveolens | |
| 0.00041 | - |
geranyl diphosphate | recombinant wild-type enzyme, pH and temperature not specified in the publication | Nicotiana suaveolens | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 1.45 | - |
geranyl diphosphate | recombinant mutant F266T, pH and temperature not specified in the publication | Nicotiana suaveolens | |
| 2 | - |
geranyl diphosphate | recombinant mutant F266V, pH and temperature not specified in the publication | Nicotiana suaveolens | |
| 2.157 | - |
geranyl diphosphate | recombinant wild-type enzyme, pH and temperature not specified in the publication | Nicotiana suaveolens | |
| 2.416 | - |
geranyl diphosphate | recombinant mutant F266S, pH and temperature not specified in the publication | Nicotiana suaveolens | |
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