Related Experiment Video
Updated: May 11, 2025
11:36
A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
10.8K
Structural Analysis and Mutability Landscape-Guided Engineering of ω-Transaminase for Improved Catalytic Performance
Puhong Yi1,2,3, Yue Xu1,2,3, Yuhua Hao1,2,3
1The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, P. R. China.
Journal of Agricultural and Food Chemistry
|April 17, 2025
Summary
ω-Transaminases (ω-TAs) are key biocatalysts for chiral amine synthesis. Engineering a Salmonella enterica ω-TA (SeTA) via distal mutation I22D significantly enhanced its activity and stability for producing the herbicide l-phosphinothricin (l-PPT).
Area of Science:
- Biocatalysis
- Enzyme Engineering
- Organic Synthesis
Background:
- ω-Transaminases (ω-TAs) are crucial biocatalysts for asymmetric synthesis of chiral amines.
- Salmonella enterica ω-TA (SeTA) is a promising enzyme for producing the herbicide l-phosphinothricin (l-PPT).
- Improving SeTA's catalytic activity and stability is essential for its industrial application.
Purpose of the Study:
- To enhance the catalytic activity and stability of SeTA.
- To investigate the structural and mechanistic basis for improved enzyme performance.
- To provide insights for engineering other ω-TAs.
Main Methods:
- Structural analysis and mutability landscape construction of SeTA.
- Site-directed mutagenesis to create distal mutants.
- Enzyme activity assays and thermal stability measurements (half-life, Tm).
- Molecular dynamics (MD) simulations to elucidate allosteric effects.
Main Results:
- A flexible loop1 region was identified as important for catalytic activity.
- The distal mutant I22D (M1) exhibited a 3.24-fold increase in specific activity.
- M1 demonstrated significantly enhanced thermal stability, with longer half-lives at 45°C and 55°C, and a higher melting temperature (Tm).
- MD simulations revealed that the I22D mutation induced long-range allosteric effects, altering the active site pocket and substrate tunnel.
Conclusions:
- Distal mutations can effectively enhance ω-TA performance through allosteric mechanisms.
- The engineered SeTA mutant shows improved catalytic efficiency and stability.
- This study offers valuable insights for the rational design of ω-TAs for industrial applications.
Related Concept Videos
01:07Catalytically Perfect Enzymes
3.8K
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.
Most enzymes...
Most enzymes...
3.8K
01:13Introduction to Mechanisms of Enzyme Catalysis
7.8K
For many years, scientists thought that enzyme-substrate binding took place in a simple "lock-and-key" fashion. This model stated that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view scientists call induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes...
7.8K
01:32Chemiosmosis
96.2K
Oxidative phosphorylation is a highly efficient process that generates large amounts of adenosine triphosphate (ATP), the basic unit of energy that drives many cellular processes. Oxidative phosphorylation involves two processes— the electron transport chain and chemiosmosis.
Electron Transport Chain
The electron transport chain involves a series of protein complexes on the inner mitochondrial membrane that undergo a series of redox reactions. At the end of this chain, the electrons...
Electron Transport Chain
The electron transport chain involves a series of protein complexes on the inner mitochondrial membrane that undergo a series of redox reactions. At the end of this chain, the electrons...
96.2K
01:19Turnover Number and Catalytic Efficiency
9.7K
The turnover number of an enzyme is the maximum number of substrate molecules it can transform per unit time. Turnover numbers for most enzymes range from 1 to 1000 molecules per second. Catalase has the known highest turnover number, capable of converting up to 2.8×106 molecules of hydrogen peroxide into water and oxygen per second. Lysozyme has the lowest known turnover number of half a molecule per second.
Chymotrypsin is a pancreatic enzyme that breaks down proteins during digestion....
Chymotrypsin is a pancreatic enzyme that breaks down proteins during digestion....
9.7K
01:19Allosteric Proteins-ATCase
5.6K
Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
5.6K
01:48ATP Synthase: Mechanism
13.6K
In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased...
13.6K