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THERMOPHILIC OLD YELLOW ENZYME: STRUCTURE AND KINETIC CHARACTERISATION

Adalbjornsson, Bjorn

[Thesis]. Manchester, UK: The University of Manchester; 2012.

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Abstract

The Old Yellow Enzyme (OYE) family of enzymes has been shown to reduce industrially important chemicals and has been used to study quantum tunnelling during enzymatic hydrogen transfer. Though extensively studied, only mesophilic homologues have been studied within the enzyme family. This thesis discusses the characterisation of Thermophilic Old Yellow Enzyme (TOYE), from Thermoanaerobacter pseudethanolicus, and provides the first published crystal structure of a thermophilic OYE-family member. In addition to increased thermostability compared to mesophilic homologues, thermophilic enzymes are important for use in industrial as often they are more stable towards organic solvents used in industry than their mesophilic homologues while catalysing the same reactions. This makes thermophiles and hyperthermophiles interesting targets for investigating the importance of enzyme dynamics during catalysis. They have also been used to study the linking of protein motion to quantum tunnelling during hydrogen transfer in other enzyme systems. In the work for this thesis, the basic characteristics of TOYE were examined. Thermal stability up to 70 °C was shown by CD and fluorescence studies and the preference towards reductive coenzyme was analysed by stopped-flow studies. Structural studies were conducted using X-ray crystallography, electron microscopy and sedimentation velocity studies. The crystal structure revealed a tetrameric enzyme with a relatively large active site. Evidence for higher oligomeric states was also obtained. The potential use of TOYE as a biocatalyst was explored by steady-state reaction, biotransformation and organic solvent resistance studies. The temperature dependences of kinetic isotope effects were used to examine the presence of tunnelling and importance active site geometry during catalysis and compared to previously described enzymes. These studies introduce a new and unique OYE-family member, allowing for more in-depth analysis of TOYE.