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Updated: May 9, 2025

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
Functional and Mechanistic Insights into the Fatty-Acid CoA Ligase FadK in Escherichia coli
Dafeng Liu1,2, Ablikim Abdiriyim1, Lvxia Zhang1
1Xinjiang Key Laboratory of Lavender Conservation and Utilization, College of Biological Sciences and Technology, Yili Normal University, 835000 Yining, Xinjiang, China.
Background:
Escherichia coli (E. coli) is a common opportunistic bacterial pathogen in both human and animal populations. Fatty acids serve as the central carbon and energy source, a process mediated by fatty acid-coenzyme A (CoA) ligases encoded by fad genes such as FadK. However, the function and the mechanism of FadK remain unclear.
Methods:
The three-dimensional structure of FadK was modeled using AlphaFold2. After expression and purification, monomeric FadK was successfully isolated. The enzymatic activity was assayed, and real-time quantitative polymerase chain reaction (RT-qPCR) was performed to quantify FadK expression levels.
Results:
In enzymatic assays of fatty acid CoA ligase activity, caprylic acid was found to be the optimal substrate for FadK. We determined the optimal catalytic conditions for FadK, which include a pH of 7.4, ATP concentration of 0.6 mM, CoA concentration of 0.8 mM, and Mg2+ concentration of 0.8 mM at 37 °C. Notably, the activity of FadK showed a decrease with increasing concentrations of dodecyl-AMP, which was further confirmed by the RT-qPCR results.
Conclusions:
Our findings will serve as a fundamental framework for the development of innovative therapeutics that target E. coli infections.
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