Mechanisms of enzyme adaptation to extreme environments: The rational design of a thermophilic chorismate mutase
ForfatterWilkins, Ryan Scott
Extremophiles, especially those living in high-temperature environments, exhibit unique enzymatic mechanisms that allow survival under conditions detrimental to most life forms. Of particular interest is thermophiles, which are able to thrive at temperatures at which psychrophiles and mesophiles unfold and cease to function. The mechanisms by which these enzymes are able to overcome this temperature extreme are not yet well understood, leading to an increasing interest in studying how they function. Thermophiles have a multitude of applications under extreme conditions in industrial processes such as pharmaceuticals, waste management, and textiles, so a more complete understanding of these molecular mechanisms can lead to the development of novel enzymes for these purposes.
This doctoral thesis focuses on exploring the molecular adaptations of the enzyme chorismate mutase (CM). Chorismate mutases are found in bacteria and plants where they catalyze the conversion of chorismate to prephenate, an important precursor in the biosynthesis of aromatic amino acids. In this thesis, I characterize a new mesophilic chorismate mutase from B. pumilus, and a new thermophilic chorismate mutase from an unknown organism found from bioprospect soils in Antarctica. Using the thermophile as a guide, I attempt to mutate the mesophilic CM, inducing thermophilic behavior.
The research in this thesis employs empirical valence bond (EVB) simulations to elucidate the molecular behavior of chorismate mutase. The methodology involves detailed computational modeling, to obtain accurate free energy estimates of the enzymatic reaction, emphasizing the enzyme’s structural and functional adaptations to different environments. By conducting simulations across a range of temperatures, I am furthermore able to extract the enthalpic and entropic contributions to the activation free energy, providing insights into the molecular dynamics and stability mechanisms of chorismate mutase. This enables highlighting significant differences in enzyme behavior between normal and extreme environmental conditions.
The findings contribute to a deeper understanding of enzyme adaptation mechanisms in extremophiles. The study also discusses the potential of EVB simulations as a powerful tool for exploring enzyme behavior in extremophiles, setting the stage for future research in this field. The thesis concludes by underscoring the importance of computational approaches in advancing our understanding of life under extreme conditions and their practical applications in industry.
Paper I: Wilkins, R.S., Lund, B.A., Isaksen, G.V., Åqvist, J. & Brandsdal, B.O. (2024). Accurate Computation of Thermodynamic Activation Parameters in the Chorismate Mutase Reaction from Empirical Valence Bond Simulations. Journal of Chemical Theory and Computation, 20(1), 451–458. Also available in Munin at https://hdl.handle.net/10037/32710.
Paper II: Wilkins, R.S., Lund, B.A., Isaksen, G.V., Åqvist, J. & Brandsdal, B.O. Biophysical characterization and analysis of a mesophilic chorismate mutase from B. pumilus. (Manuscript). Pre-print available on bioRxiv at https://doi.org/10.1101/2023.04.20.537678.
Paper III: Wilkins, R.S., Skagseth, S., Williamson, A., Lund, B.A., Åqvist, J. & Brandsdal, B.O. Characterization of a thermophilic chorismate mutase and its utility as a template in rational enzyme design. (Manuscript).
ForlagUiT Norges arktiske universitet
UiT The Arctic University of Norway
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