Related Experiment Video
Updated: Jun 20, 2025
08:37
A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
Published on: April 30, 2018
7.6K
Single Point Mutation Abolishes Water Capture in Germacradien-4-ol Synthase.
Víctor González Requena1, Prabhakar L Srivastava1, David J Miller1
1School of Chemistry, Main Building, Cardiff University, Park Place, Cardiff, CF10 3AT, United Kingdom.
Chembiochem : a European Journal of Chemical Biology
|July 20, 2024
Summary
Researchers modified a sesquiterpene cyclase enzyme (GdolS) by altering specific amino acids. This engineering controls hydroxylation, enabling tailored biocatalysis for producing valuable oxygenated sesquiterpenoids.
Area of Science:
- Biochemistry
- Enzyme Engineering
- Organic Chemistry
Background:
- Sesquiterpene cyclases (SgS) synthesize diverse sesquiterpenoids from farnesyl diphosphate.
- The high-fidelity (-)-germacradien-4-ol synthase (GdolS) produces (-)-germacradien-4-ol.
- Understanding enzyme mechanisms is crucial for biocatalysis and synthetic biology.
Purpose of the Study:
- To investigate the role of specific amino acid residues in the water control mechanism of GdolS.
- To explore how mutations affect the hydroxylation of sesquiterpenoid products.
- To provide insights for engineering sesquiterpene synthases for targeted biocatalytic production.
Main Methods:
- Site-directed mutagenesis of GdolS, focusing on Ala176 and His150.
- Analysis of enzymatic products using biochemical assays.
- Characterization of modified enzyme activity and product profiles.
Main Results:
- Replacing Ala176 with non-polar residues abolished hydroxylation, yielding germacrene A and D.
- Replacing Ala176 with polar residues maintained hydroxylation.
- His150 was not essential for water addition but likely aids carbocation stabilization.
Conclusions:
- Key residues, particularly Ala176, control the hydroxylation activity of GdolS.
- Engineering GdolS can redirect its catalytic activity towards desired oxygenated sesquiterpenoids.
- This research facilitates the design of novel biocatalysts for efficient sesquiterpenoid production.
More Related Videos
Related Concept Videos
01:39Mutations
81.6K
Overview
81.6K
01:16In-vitro Mutagenesis
13.9K
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
13.9K
02:46Genome Copying Errors
4.2K
DNA replication is a well-evolved process that copies millions of base pairs with high fidelity during each cell division. Occasionally a wrong base or a long stretch of wrong bases may get added to the daughter strands. If the errors are left unchecked, cells might accumulate several mutations that might endanger their survival. Therefore, the copying errors are checked and repaired at three levels.
4.2K
02:08Gene Conversion
9.7K
Other than maintaining genome stability via DNA repair, homologous recombination plays an important role in diversifying the genome. In fact, the recombination of sequences forms the molecular basis of genomic evolution. Random and non-random permutations of genomic sequences create a library of new amalgamated sequences. These newly formed genomes can determine the fitness and survival of cells. In bacteria, homologous and non-homologous types of recombination lead to the evolution of new...
9.7K