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Literature summary extracted from

  • Rinkel, J.; Lauterbach, L.; Rabe, P.; Dickschat, J.
    Two diterpene synthases for spiroalbatene and cembrene A from allokutzneria albata (2018), Angew. Chem. Int. Ed. Engl., 57, 3238-3241 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.3.B68 phylogenetic tree, recombinant expression of wild-type and mutant enzymes in Escherichia coli Allokutzneria albata
4.2.3.150 phylogenetic tree, recombinant expression of wild-type and mutant enzymes in Escherichia coli Allokutzneria albata

Protein Variants

EC Number Protein Variants Comment Organism
4.2.3.B68 E179K site-directed mutagenesis, inactive mutant Allokutzneria albata
4.2.3.B68 E179Q site-directed mutagenesis, the mutation results in strongly reduced activity with both metal cations compared to wild-type Allokutzneria albata
4.2.3.B68 E193D site-directed mutagenesis, the mutation results in reduced activity with Mg2+ compared to wild-type, or a complete loss of activity with Mn2+ Allokutzneria albata
4.2.3.B68 L72A site-directed mutagenesis, the mutant variant of enzyme SaS gives no soluble protein Allokutzneria albata
4.2.3.B68 P201T site-directed mutagenesis, the mutant variant of SaS shows significantly reduced activity with both metal cofactors compared to wild-type Allokutzneria albata
4.2.3.B68 P65A site-directed mutagenesis, the mutation yields wild-type levels of enzyme, but the activity drops significantly with Mg2+ and Mn2+ Allokutzneria albata
4.2.3.B68 Q160E site-directed mutagenesis, the mutant enzyme is well expressed, but shows no activity with either Mg2+ or Mn2+ Allokutzneria albata
4.2.3.B68 R145K site-directed mutagenesis, the mutation results in reduced activity with Mg2+ compared to wild-type, or a complete loss of activity with Mn2+ Allokutzneria albata
4.2.3.B68 R145M site-directed mutagenesis, the mutant variant shows only a slight decrease in activity for both metal cofactors compared to wild-type Allokutzneria albata
4.2.3.150 D212E site-directed mutagenesis, the mutation results in strongly decreased activity with Mg2+ and Mn2+ Allokutzneria albata

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
4.2.3.B68 Mg2+ activates Allokutzneria albata
4.2.3.B68 Mn2+ activates Allokutzneria albata
4.2.3.B68 additional information influence of the length of this salt bridge, likely by alteration of the tilt angle between helices G and F on the active-site architecture, with consequences for metal binding. A short salt bridge with participation of Asp seems to favor catalysis with Mn2+ while a long salt bridge with Glu fosters Mg2+ binding Allokutzneria albata
4.2.3.150 Mg2+ activates Allokutzneria albata
4.2.3.150 Mn2+ activates Allokutzneria albata

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.3.B68 geranylgeranyl diphosphate Allokutzneria albata
-
 spiroalbatene + diphosphate
-
 ?
4.2.3.B68 geranylgeranyl diphosphate Allokutzneria albata DSM 44149
-
 spiroalbatene + diphosphate
-
 ?
4.2.3.B68 additional information Allokutzneria albata for spiroalbatene synthase, the pH- and Mn2+-dependent formation of the side product thunbergol is observed, which is biosynthetically linked to spiroalbatene ?
-
 ?
4.2.3.B68 additional information Allokutzneria albata DSM 44149 for spiroalbatene synthase, the pH- and Mn2+-dependent formation of the side product thunbergol is observed, which is biosynthetically linked to spiroalbatene ?
-
 ?
4.2.3.150 geranylgeranyl diphosphate Allokutzneria albata
-
 (S)-(+)-cembrene A + diphosphate
-
 ?
4.2.3.150 geranylgeranyl diphosphate Allokutzneria albata DSM 44149
-
 (S)-(+)-cembrene A + diphosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
4.2.3.B68 Allokutzneria albata
-
 isolated from tropical soil
-
4.2.3.B68 Allokutzneria albata DSM 44149
-
 isolated from tropical soil
-
4.2.3.150 Allokutzneria albata
-
 isolated from tropical soil
-
4.2.3.150 Allokutzneria albata DSM 44149
-
 isolated from tropical soil
-

Reaction

EC Number Reaction Comment Organism Reaction ID
4.2.3.B68 geranylgeranyl diphosphate = spiroalbatene + diphosphate cyclization mechanism from GGPP to spiroalbatene involving 1,14-cyclization to cation, followed by a 1,2-hydride migration, another 1,2-hydride shift, and ring closure, followed by a third 1,2-hydride shift and cyclization, yielding a cyclopropane intermediate, and opening of the three-membered ring with concomitant ring closure Allokutzneria albata
a
4.2.3.150 geranylgeranyl diphosphate = (R)-cembrene A + diphosphate the cyclization mechanism from geranylgeranyl diphosphate to cembrene A requires a 1,14-cyclization to the ent-cembranyl cation and deprotonation of one of the geminal methyl groups, reaction mechanism, overview Allokutzneria albata
a

Source Tissue

EC Number Source Tissue Comment Organism Textmining
4.2.3.B68 additional information the enzyme seems to be not expressed in laboratory cultures Allokutzneria albata
-
4.2.3.150 additional information the enzyme seems to be not expressed in laboratory cultures Allokutzneria albata
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.3.B68 geranylgeranyl diphosphate
-
 Allokutzneria albata spiroalbatene + diphosphate
-
 ?
4.2.3.B68 geranylgeranyl diphosphate
-
 Allokutzneria albata DSM 44149 spiroalbatene + diphosphate
-
 ?
4.2.3.B68 additional information for spiroalbatene synthase, the pH- and Mn2+-dependent formation of the side product thunbergol is observed, which is biosynthetically linked to spiroalbatene Allokutzneria albata ?
-
 ?
4.2.3.B68 additional information the defined stereochemical anchors at the deuterated carbon atoms are used to solve the absolute configuration of spiroalbatene by determining the relative configurations of the stereospecifically deuterated products. Enzyme SaS can potentially yield ent-(S)-(+)-cembrene A from intermediate A.. Product identification by NMR spectroscopic analysis. Development of a method that uses the stereoselectively deuterated precursors (S)- and (R)-(1-13C,1-2H)FPP and (S)- and (R)-(1-13C,1-2H)GPP to determine the absolute configurations of terpenes Allokutzneria albata ?
-
 ?
4.2.3.B68 additional information for spiroalbatene synthase, the pH- and Mn2+-dependent formation of the side product thunbergol is observed, which is biosynthetically linked to spiroalbatene Allokutzneria albata DSM 44149 ?
-
 ?
4.2.3.B68 additional information the defined stereochemical anchors at the deuterated carbon atoms are used to solve the absolute configuration of spiroalbatene by determining the relative configurations of the stereospecifically deuterated products. Enzyme SaS can potentially yield ent-(S)-(+)-cembrene A from intermediate A.. Product identification by NMR spectroscopic analysis. Development of a method that uses the stereoselectively deuterated precursors (S)- and (R)-(1-13C,1-2H)FPP and (S)- and (R)-(1-13C,1-2H)GPP to determine the absolute configurations of terpenes Allokutzneria albata DSM 44149 ?
-
 ?
4.2.3.150 geranylgeranyl diphosphate
-
 Allokutzneria albata (S)-(+)-cembrene A + diphosphate
-
 ?
4.2.3.150 geranylgeranyl diphosphate
-
 Allokutzneria albata DSM 44149 (S)-(+)-cembrene A + diphosphate
-
 ?
4.2.3.150 additional information product identification by NMR spectroscopic analysis. Development of a method that uses the stereoselectively deuterated precursors (S)- and (R)-(1-13C,1-2H)farnesyl diphosphate and (S)- and (R)-(1-13C,1-2H)geranyl diphosphate to determine the absolute configurations of terpenes Allokutzneria albata ?
-
 ?
4.2.3.150 additional information product identification by NMR spectroscopic analysis. Development of a method that uses the stereoselectively deuterated precursors (S)- and (R)-(1-13C,1-2H)farnesyl diphosphate and (S)- and (R)-(1-13C,1-2H)geranyl diphosphate to determine the absolute configurations of terpenes Allokutzneria albata DSM 44149 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
4.2.3.B68 SAS
-
 Allokutzneria albata
4.2.3.B68 spiroalbatene synthase
-
 Allokutzneria albata
4.2.3.150 CAS
-
 Allokutzneria albata

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.3.B68 7.4 8.2 assay at Allokutzneria albata

General Information

EC Number General Information Comment Organism
4.2.3.B68 metabolism spiroalbatene may be oxidized to an unknown product by a genetically clustered cytochrome P450 Allokutzneria albata