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Title: Natural evolution and engineering of transketolase
Author: Costelloe, S. J.
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2006
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Transketolase (TK) is an important metabolic enzyme in all organisms. The enantioselective carbon-carbon bond forming action of TK makes it significantly interesting for biocatalysis. TKs from different organisms exhibit varied substrate specificities mediated by a small number of differing residues, giving insight into a potential route for engineering new enzymes. TK uses Thiamine Pyrophosphate (TPP) as cofactor, as do many other evolutionarily related enzymes. In Chapter 3, a phylogenetic analysis of the catalytic domains of TPP-dependent enzymes enabled the assembly of the evolutionary history of this enzyme family. In Chapter 4, the evolution of the differing substrate specificities of Eschericia coli TK and Saccharomyces cerevisiae TK from their most recent common ancestor enzyme was analysed. A detailed phylogenetic analysis of TK was performed, yielding the amino acid sequences of the ancient TKs. TKs linking the common ancestor of E. coli and S.cerevisiae with extant E. coli TK were "resurrected" and assayed for the P-Hydroxypyruvate (P-HPA) + glycoaldehyde (GA) reaction. The common ancestor TK and E. coli TK were assayed for many reactions to define their substrate repertoires and to elucidate any evolutionary trends in substrate specificity. p-HPA is the ideal donor substrate for TK, since it yields CO2, making reactions irreversible. p-HPA is not readily available commercially and is very expensive. To be industrially viable, a cheaper donor is needed. Pyruvate is much cheaper than P-HPA yet TK has never been shown to use pyruvate as a donor. Chapter 5 describes a comparison of TK with the pyruvate utilising TPP-dependent enzymes DXPS and PDC, suggesting residues which may confer pyruvate usage. Mutants were generated and tested for activity with pyruvate. In Chapter 6, the non-catalytic C-terminal domain of TK (TKC domain) was examined. The function of the TKC domain is currently undefined. In this chapter the TKC domain is removed and it is shown that the TK enzyme activity is retained.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available