Reaction #677866

ord-9fbb8acc37d447e4993cf7e0370f5436

Reaction equation

O=S(=O)(O)CCN1CCN(CCO)CC1
HEPES
CC(C)=CCC/C(C)=C/CC/C(C)=C/CC/C(C)=C/COP(=O)([O-])OP(=O)([O-])[O-]
GGPP
[Cl-].[Cl-].[Mg+2]
MgCl2
[Cl-].[Cl-].[Mg+2]
MgCl2
CC(C)=CCC/C(C)=C\CC/C(C)=C\COP(=O)([O-])OP(=O)([O-])[O-]
2Z-6Z-farnesyl diphosphate
O[C@H](CS)[C@H](O)CS
DTT
CC(C)=CCC/C(C)=C\COP(=O)([O-])OP(=O)([O-])[O-]
NDP
[Cl-].[K+]
KCl
CC(C)=CCC/C(C)=C/COP(=O)([O-])OP(=O)([O-])[O-]
GPP
CC(C)=CCC/C(C)=C/CC/C(C)=C/COP(=O)([O-])OP(=O)([O-])[O-]
E-E-farnesyl diphosphate
→
CC(C)=CCC[C@H](C)[C@@H]1C=CC(C)=CC1
Zingiberene

Reactants

—
teflon
O=S(=O)(O)CCN1CCN(CCO)CC1
HEPES
CC(C)=CCC/C(C)=C/CC/C(C)=C/CC/C(C)=C/COP(=O)([O-])OP(=O)([O-])[O-]
GGPP
[Cl-].[Cl-].[Mg+2]
MgCl2
[Cl-].[Cl-].[Mg+2]
MgCl2
CC(C)=CCC/C(C)=C\CC/C(C)=C\COP(=O)([O-])OP(=O)([O-])[O-]
2Z-6Z-farnesyl diphosphate
O[C@H](CS)[C@H](O)CS
DTT
CC(C)=CCC/C(C)=C\COP(=O)([O-])OP(=O)([O-])[O-]
NDP
[Cl-].[K+]
KCl
CC(C)=CCC/C(C)=C/COP(=O)([O-])OP(=O)([O-])[O-]
GPP
CC(C)=CCC/C(C)=C/CC/C(C)=C/COP(=O)([O-])OP(=O)([O-])[O-]
E-E-farnesyl diphosphate

Reagents

None

Solvents

—
glass
OCC(O)CO
glycerol

Conditions

Temperature
30°CELSIUS
Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    Othercrimp cap immediately

Procedure

Activity assays were performed in 20 mL glass vials in a total volume of 500 μL 50 mM HEPES, pH7.2, 100 mM KCl, 7.5 mM MgCl2, 20 μM MgCl2, 5% (v/v) glycerol, 5 mM DTT with 50 μL protein and either 2 mM cis-FPP (2Z-6Z-farnesyl diphosphate), trans-FPP (E-E-farnesyl diphosphate), GPP (geranyl diphosphate), NDP (neryl diphosphate), or GGPP (geranylgeranyl diphosphate) as a substrate (Echelon Biosciences Incorporated, Salt Lake City, USA). Vials were closed with a teflon lined crimp cap immediately and incubated under moderate shaking for 1 hour at 30° C.

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US09234193B2uspto-grants-2016_01