Cloned (Comment) | Organism |
---|---|
recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain C41 OverExpress | Salvia sclarea |
Protein Variants | Comment | Organism |
---|---|---|
N431F | site-directed mutagenesis, the mutant predominantly produces manool epimers with stereoselectivity of 90% and 60-62%, respectively | Salvia sclarea |
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 |
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 |
N431Y | site-directed mutagenesis, the mutant predominantly produces manool epimers with stereoselectivity of 90% and 60-62%, respectively | Salvia sclarea |
S433C | site-directed mutagenesis, the mutation has no effect on product outcome | Salvia sclarea |
T436C | site-directed mutagenesis, the mutation has no effect on product outcome | Salvia sclarea |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.006 | - |
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate | recombinant enzyme mutant N431I, pH 7.2, 30°C | Salvia sclarea | |
0.007 | - |
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate | recombinant wild-type enzyme, pH 7.2, 30°C | Salvia sclarea | |
0.01 | - |
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate | recombinant enzyme mutant N431Q, pH 7.2, 30°C | Salvia sclarea |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate + H2O | Salvia sclarea | i.e. 8alpha-hydroxy-copalyl diphosphate | (13R)-sclareol + diphosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Salvia sclarea | K4HYB0 | - |
- |
Purification (Comment) | Organism |
---|---|
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 | Comment | Organism | Reaction ID |
---|---|---|---|
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate + H2O = sclareol + diphosphate | via a 13-yl-carbocation | Salvia sclarea |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate + H2O | i.e. 8alpha-hydroxy-copalyl diphosphate | Salvia sclarea | (13R)-sclareol + diphosphate | - |
? | |
(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 | - |
? | |
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 | Comment | Organism |
---|---|---|
SsSS | - |
Salvia sclarea |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Salvia sclarea |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.38 | - |
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate | recombinant enzyme mutant N431I, pH 7.2, 30°C | Salvia sclarea | |
0.47 | - |
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate | recombinant enzyme mutant N431Q, pH 7.2, 30°C | Salvia sclarea | |
0.53 | - |
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate | recombinant wild-type enzyme, pH 7.2, 30°C | Salvia sclarea |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.2 | - |
assay at | Salvia sclarea |
General Information | Comment | Organism |
---|---|---|
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 |
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 |
additional information | homology modeling | Salvia sclarea |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
47 | - |
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate | recombinant enzyme mutant N431Q, pH 7.2, 30°C | Salvia sclarea | |
63.3 | - |
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate | recombinant enzyme mutant N431I, pH 7.2, 30°C | Salvia sclarea | |
75.7 | - |
(13E)-8alpha-hydroxylabd-13-en-15-yl diphosphate | recombinant wild-type enzyme, pH 7.2, 30°C | Salvia sclarea |