Related Experiment Video
Updated: Aug 8, 2026
10:50
Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
Conversion of hydroxyphenylpyruvate dioxygenases into hydroxymandelate synthases by directed evolution
Helen M O'Hare1, Fanglu Huang, Andrew Holding
1Department of Chemistry, University of Cambridge, UK.
FEBS Letters
|May 30, 2006
Summary
Directed evolution readily converted hydroxyphenylpyruvate dioxygenase (HppD) to hydroxymandelate synthase (HmaS) activity with two mutations. Engineering HppD activity into HmaS was unsuccessful, indicating higher catalytic demands for homogentisate production.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Hydroxymandelate synthase (HmaS) and hydroxyphenylpyruvate dioxygenase (HppD) are non-heme iron-dependent dioxygenases.
- Both enzymes share a common substrate and initial catalytic step, but diverge to produce distinct products: hydroxymandelate for secondary metabolism and homogentisate for tyrosine catabolism.
Purpose of the Study:
- To investigate the structural and functional differences between HmaS and HppD active sites.
- To explore the possibility of interconverting their enzymatic activities through directed evolution.
Main Methods:
- Utilized directed evolution techniques to engineer HmaS and HppD.
- Introduced specific amino acid mutations to alter enzyme function.
Main Results:
- Successfully engineered HppD to exhibit HmaS activity through only two amino acid substitutions.
- Failed to engineer HmaS to exhibit HppD activity, suggesting significant differences in catalytic requirements.
Conclusions:
- The active site of HppD can be readily modified to adopt HmaS function.
- Homogentisate synthesis by HppD imposes greater chemical and steric constraints on the active site compared to hydroxymandelate synthesis by HmaS.
More Related Videos
Related Concept Videos
01:24Evolution of New Traits in Microbes
Microorganisms evolve rapidly due to their large population sizes and short generation times, often exhibiting measurable changes within days under laboratory conditions. Natural selection acts on standing genetic variation, enabling the retention and amplification of beneficial traits that confer fitness advantages in changing environments.Adaptive Pigment Regulation in RhodobacterIn Rhodobacter, a genus of purple non-sulfur bacteria, light-harvesting pigments such as bacteriochlorophyll and...
02:44Oxidation of Alkenes: Syn Dihydroxylation with Osmium Tetraoxide
Alkenes are converted to 1,2-diols or glycols through a process called dihydroxylation. It involves the addition of two hydroxyl groups across the double bond with two different stereochemical approaches, namely anti and syn. Dihydroxylation using osmium tetroxide progresses with syn stereochemistry.
01:24Diels–Alder Reaction: Characteristics of Dienophiles
In a Diels–Alder reaction, the diene is usually an electron-rich system and acts as a nucleophile, whereas the dienophile is electron-deficient and functions as an electrophile. Much like the diene, the nature of the dienophile significantly impacts the outcome of the reaction.
Characteristics of Dienophiles
Generally, the best dienophiles are alkenes containing electron-withdrawing substituents such as carbonyl, nitrile, and nitro groups. The feasibility of a Diels–Alder reaction depends on...
Characteristics of Dienophiles
Generally, the best dienophiles are alkenes containing electron-withdrawing substituents such as carbonyl, nitrile, and nitro groups. The feasibility of a Diels–Alder reaction depends on...
01:22Bioreactor Controls-III
Strain improvement is a foundational strategy in industrial microbiology aimed at maximizing microbial productivity, particularly because natural isolates typically yield commercially valuable products in very low concentrations. Although optimizing the culture medium and environmental conditions can improve yields, these adjustments are inherently limited by the organism’s genetic potential. As a result, the focus shifts toward genetic modifications to enhance biosynthetic capacity. The...
03:08Hydroboration-Oxidation of Alkenes
In addition to the oxymercuration–demercuration method, which converts the alkenes to alcohols with Markovnikov orientation, a complementary hydroboration-oxidation method yields the anti-Markovnikov product. The hydroboration reaction, discovered in 1959 by H.C. Brown, involves the addition of a B–H bond of borane to an alkene giving an organoborane intermediate. The oxidation of this intermediate with basic hydrogen peroxide forms an alcohol.
01:07Catalytically Perfect Enzymes
The theory of catalytically perfect enzymes was first proposed by W.J. Albery and J. R. Knowles in 1976. These enzymes catalyze biochemical reactions at high-speed. Their catalytic efficiency values range from 108-109 M-1s-1. These enzymes are also called 'diffusion-controlled' as the only rate-limiting step in the catalysis is that of the substrate diffusion into the active site. Examples include triose phosphate isomerase, fumarase, and superoxide dismutase.