Mechanistic Studies of Escherichia Coli Transketolase

Abstract

The enzyme transketolase is found in nature as part of the Pentose Phosphate Pathway to rearrange large sugar phosphates. It also is an important enzyme for carboncarbon bond formation for industrial biocatalysis. The work presented in this thesis describes the purification, crystallisation, characterisation and structural determination of the recombinant Escherichia coli transketolase complexed with the substrate hydroxypyruvate and potential inhibitor fluoropyruvate. The native transketolase and the transketolase-hydroxypyruvate structures were solved to a 1.18 and 1.05 Å resolution respectively. The transketolase structures show a chain of ordered water molecules spanning a distance of 20 Å between the two active sites. The water molecules are linked via a network of hydrogen bonds and they are proposed to facilitate proton transfer between the two-thiamine pyrophosphate molecules, thereby providing a method of communication between the two active sites of the enzyme. The transketolase-hydroxypyruvate structure shows the hydroxypyruvate substrate forming a covalent bond to the thiamine pyrophosphate thereby creating a a,b-dihydroxyethyl–thiamine pyrophosphate complex within the enzyme active site. The novel transketolase-fluoropyruvate structure solved to a 1.60 Å resolution, it produced a snapshot image of the ketol donor prior to formation of the active enamine intermediate. The trapped fluoropyruvate molecule is shown to form an angle that varies from the accepted Burgi-Dunitz angle of 109.5° for nucleophilic attack. However, this is inconclusive due to the low occupancy of the fluoropyruvate. In addition, kinetic studies were performed on the recombinant E. coli transketolase to investigate the inhibitory role of fluoropyruvate during the enzymatic reaction. The active site recombinant E. coli transketolase mutants H26Y and D469Y have been also been purified and characterised. The mutant H26Y complexed with fluoropyruvate was crystallised and its structure determined to 1.66 Å resolution. This structure has given an insight into why this mutation results in the formation of the opposite D-enantiomer of erythrulose rather than the L-erythrulose produced by the wild-type transketolase enzyme. The thesis also includes the purification, crystallisation, characterisation and Xray diffraction studies of the commercially useful oxygenating enzyme, 2,5- diketocamphane 1,2-monooxygenase from Pseudomonas putida. The recombinant dimeric oxygenase component of this enzyme has been crystallised and its structure solved to 1.4 Å resolution.