Characterization and Engineering of the 'Biocatalytic' Transketolase from Geobacillus stearothermophilus
by Camilla Leogrande
Date of Examination:2025-01-27
Date of issue:2025-04-24
Advisor:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Ricardo A. Mata
Persistent Address:
http://dx.doi.org/10.53846/goediss-11226
Files in this item
Name:Dissertation_Leogrande_corrected.pdf
Size:88.4Mb
Format:PDF
The following license files are associated with this item:
Abstract
English
Biocatalysis is the use of living organisms or their components to speed up chemical reactions, and is a powerful tool to enhance efficiency and sustainability in manufacturing processes. Carboligation, the formation of carbon-carbon bonds, is a critical but challenging reaction to achieve. To date, carboligating enzymes remain underrepresented among commercially employed biocatalysts. Enzymes utilizing the organic cofactor thiamine diphosphate (ThDP) are particularly promising for addressing this gap and have been extensively investigated. The specific chemistry they catalyze, in fact, enables the coupling of two carbon atoms with similar polarity, in particular when both are electrophilic in nature. Transketolases are members of the superfamily of ThDPdependent enzymes. Their physiological substrates are phosphorylated sugars, but they have been widely engineered to expand their substrate scope to include non-phosphorylated sugars, as well as aliphatic and aromatic compounds. The transketolase from Geobacillus stearothermophilus (GstTK) was the first thermostable transketolase to be cloned and evaluated for its synthetic potential. In this work, we present a comprehensive characterization of the wild type enzyme to assess and improve its industrial applicability. Our analysis encompasses the elucidation of the structure and the investigation of the origins of the enzyme’s thermostability. In particular, we uncover the presence of higher oligomeric states beyond the functional dimer observed in most transketolases. We report the negative correlation of the larger oligomers with both activity and thermostability, thus suggesting that strategies aimed at reducing or eliminating these oligomers could enhance the overall enzyme performance. We conducted several biophysical experiments, including the development of a novel protocol to quantify the cofactor binding affinity using isothermal titration calorimetry (ITC). These studies reveal an unprecedented degree of cooperativity in GstTK compared to other transketolases and provide evidence for semi-holo forms of the enzyme, where only one of the two active sites of the functional dimer is occupied by the cofactor. These findings contribute to challenge the broader understanding of cooperativity, a complex phenomenon observed in all ThDP-dependent enzymes but varying in magnitude across enzyme families. Finally, we characterize and newly identify several GstTK variants, along with their counterparts from the Escherichia coli orthologue, building on previously reported mutations. Using structural information, we discuss the role of these mutations in altering the substrate specificity, particularly towards aliphatic donors and rare deoxy and/or non-phosphorylated sugars. Notably, we highlight the L118I mutation as potentially beneficial regardless of the substrates used, due to its ability to induce the cofactor into a more catalytically active conformation. Lastly, we propose future directions for the engineering of GstTK, emphasizing a focus on rare sugars, for which the enzyme demonstrates good specific activities. This work advances the understanding of GstTK’s structure, function, and mutability, providing a foundation for its optimization in industrial applications.
Keywords: Biocatalysis; Trasnketolase; Thermostability; Carboligation
