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The T296V Mutant of Amorpha-4,11-diene Synthase Is Defective in Allylic Diphosphate Isomerization but Retains the Ability To Cyclize the Intermediate (3R)-Nerolidyl Diphosphate to Amorpha-4,11-diene
- Source :
- Biochemistry. 55:6599-6604
- Publication Year :
- 2016
- Publisher :
- American Chemical Society (ACS), 2016.
-
Abstract
- The T296V mutant of amorpha-4, 11-diene synthase catalyzes the abortive conversion of the natural substrate (E,E)-farnesyl diphosphate mainly into the acyclic product (E)-β-farnesene (88%) instead of the natural bicyclic sesquiterpene amorphadiene (7%). Incubation of the T296V mutant with (3R,6E)-nerolidyl diphosphate resulted in cyclization to amorphadiene. Analysis of additional mutants of amino acid residue 296 and in vitro assays with the intermediate analogue (2Z,6E)-farnesyl diphosphate as well as (3S,6E)-nerolidyl diphosphate demonstrated that the T296V mutant can no longer catalyze the allylic rearrangement of farnesyl diphosphate to the normal intermediate (3R,6E)-nerolidyl diphosphate, while retaining the ability to cyclize (3R,6E)-nerolidyl diphosphate to amorphadiene. The T296A mutant predominantly retained amorphadiene synthase activity, indicating that neither the hydroxyl nor the methyl group of the Thr296 side chain is required for cyclase activity.
- Subjects :
- 0301 basic medicine
Amorpha-4,11-diene
Allylic rearrangement
Stereochemistry
Mutant
Mutation, Missense
Stereoisomerism
Artemisia annua
010402 general chemistry
01 natural sciences
Biochemistry
Article
Gas Chromatography-Mass Spectrometry
Substrate Specificity
03 medical and health sciences
chemistry.chemical_compound
Polyisoprenyl Phosphates
Plant Proteins
Polycyclic Sesquiterpenes
Alkyl and Aryl Transferases
Molecular Structure
Bicyclic molecule
ATP synthase
biology
Chemistry
0104 chemical sciences
Diphosphates
Kinetics
030104 developmental biology
Models, Chemical
Cyclization
Biocatalysis
biology.protein
Sesquiterpenes
Cyclase activity
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 55
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....a2e0845f78bb6a8d8b964cfe695c5063
- Full Text :
- https://doi.org/10.1021/acs.biochem.6b01004