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.EPSR