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Literature summary extracted from

  • Jia, M.; O'Brien, T.E.; Zhang, Y.; Siegel, J.B.; Tantillo, D.J.; Peters, R.J.
    Changing face a key residue for the addition of water by sclareol synthase (2018), ACS Catal., 8, 3133-3137 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.2.3.141 recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain C41 OverExpress Salvia sclarea

Protein Variants

EC Number Protein Variants Comment Organism
4.2.3.141 N431F site-directed mutagenesis, the mutant predominantly produces manool epimers with stereoselectivity of 90% and 60-62%, respectively Salvia sclarea
4.2.3.141 N431I site-directed mutagenesis, the mutant exclusively produces isoabienol, resullting from direct deprotonation of the initially generated 13-yl+ intermediate at the neighboring methyl group. There is a below 10% decrease in yield for this mutant relative to wild-type SsSS in the engineered bacterial culture Salvia sclarea
4.2.3.141 N431Q site-directed mutagenesis, the mutant selectively produces epimeric (13S)-sclareol, with SsSS:N431Q exhibiting almost complete reversal of the stereoselective addition of water, i.e., while wild-type SsSS enzyme produces (13R)-sclareol in 80% enantiomeric excess (ee), SsSS:N431Q produced 70% ee of (13S)-sclareol Salvia sclarea
4.2.3.141 N431Y site-directed mutagenesis, the mutant predominantly produces manool epimers with stereoselectivity of 90% and 60-62%, respectively Salvia sclarea
4.2.3.141 S433C site-directed mutagenesis, the mutation has no effect on product outcome Salvia sclarea
4.2.3.141 T436C site-directed mutagenesis, the mutation has no effect on product outcome Salvia sclarea

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
4.2.3.141 0.006
-
 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate recombinant enzyme mutant N431I, pH 7.2, 30°C Salvia sclarea
4.2.3.141 0.007
-
 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate recombinant wild-type enzyme, pH 7.2, 30°C Salvia sclarea
4.2.3.141 0.01
-
 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate recombinant enzyme mutant N431Q, pH 7.2, 30°C Salvia sclarea

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
4.2.3.141 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate + H2O Salvia sclarea i.e. 8alpha-hydroxy-copalyl diphosphate (13R)-sclareol + diphosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
4.2.3.141 Salvia sclarea K4HYB0
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.2.3.141 recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain C41 OverExpress by nickel affinity chromatography and dialysis Salvia sclarea

Reaction

EC Number Reaction Comment Organism Reaction ID
4.2.3.141 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate + H2O = sclareol + diphosphate via a 13-yl-carbocation Salvia sclarea
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4.2.3.141 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate + H2O i.e. 8alpha-hydroxy-copalyl diphosphate Salvia sclarea (13R)-sclareol + diphosphate
-
 ?
4.2.3.141 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate + H2O i.e. 8alpha-hydroxy-copalyl diphosphate, the enzyme shows strong stereoselectivity in formation of the (13R)-epimer, residue N431 is responsible Salvia sclarea (13R)-sclareol + diphosphate
-
 ?
4.2.3.141 additional information GC-MS reaction product analysis. Molecular docking of enzyme products in to the active site of the enzyme, modeling, overview Salvia sclarea ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
4.2.3.141 SsSS
-
 Salvia sclarea

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
4.2.3.141 30
-
 assay at Salvia sclarea

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
4.2.3.141 0.38
-
 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate recombinant enzyme mutant N431I, pH 7.2, 30°C Salvia sclarea
4.2.3.141 0.47
-
 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate recombinant enzyme mutant N431Q, pH 7.2, 30°C Salvia sclarea
4.2.3.141 0.53
-
 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate recombinant wild-type enzyme, pH 7.2, 30°C Salvia sclarea

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
4.2.3.141 7.2
-
 assay at Salvia sclarea

General Information

EC Number General Information Comment Organism
4.2.3.141 malfunction mutants N431D and N431Q selectively produce (13S)-sclareol, with SsSS:N431Q exhibiting almost complete reversal of the stereoselective addition of water, i.e., while wild-type SsSS produces (13R)-sclareol in 80% enantiomeric excess (ee), SsSS:N431Q produces 70% ee of (13S)-sclareol. The single residue change N431Q essentially flips the stereochemical outcome, changing the addition of water to the si face of the double bond in (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate providing biosynthetic access to (13S)-sclareol Salvia sclarea
4.2.3.141 metabolism the enzyme substrate is derived from bicyclization of the general diterpene precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) by class II diterpene cyclase copal-8-ol diphosphate hydratase, EC 4.2.1.133 Salvia sclarea
4.2.3.141 additional information homology modeling Salvia sclarea

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
4.2.3.141 47
-
 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate recombinant enzyme mutant N431Q, pH 7.2, 30°C Salvia sclarea
4.2.3.141 63.3
-
 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate recombinant enzyme mutant N431I, pH 7.2, 30°C Salvia sclarea
4.2.3.141 75.7
-
 (13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate recombinant wild-type enzyme, pH 7.2, 30°C Salvia sclarea