Novel Insights in Structure and Mechanism of Escherichia coli Transketolase

by Fabian Rabe von Pappenheim
Doctoral thesis
Date of Examination:2017-05-23
Date of issue:2018-05-18
Advisor:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Ralf Ficner
Referee:Dr. Manfred Konrad
Persistent Address: http://dx.doi.org/10.53846/goediss-6876

 

 

Files in this item

Name:Dissertation_FvP.pdf
Size:24.6Mb
Format:PDF

The following license files are associated with this item:

Abstract

English

Thiamine diphosphate (ThDP) dependent enzymes fulfill a number of indispensable functions in metabolism. Further, they are well-researched work-horses of biotechnology. Thus, deepening of our mechanistic and structural knowledge of these is imperative in improving biotechnological approaches, gain fundamental insight in enzymological principles and possibly develop antibiotics. In this work we present fundamental insights in multiple mechanistic aspects of ThDP-dependent enzymes using the Escherichia coli transketolase as model system. Studies on the control of cofactor dynamics and their impact on catalysis provided a model by which intrinsic cofactor movement facilitates bond strain reducing the energy barrier for C-C bond cleavage. Further, a core tenet of ThDP-facilitated catalysis was challenged by generating a non-aromatic, electrophilic keto-intermediate in a transferase by mutagenesis of a single, intermediate-stabilizing residue. The role of conserved features of transketolases in controlling cofactor properties was gauged. The mode of action of the naturally occurring antibiotic 2’-methoxy thiamine diphosphate was investigated and a model based on enzyme-selective disturbance of the cofactor activation, supported by ultra-high resolution crystallographic data and NMR data, was derived.
Keywords: Enzymology; Thiamine Diphosphate; Carbohydrates; Enzyme Dynamics
 
Statistik