Chudziak, C.M.; (2007) Production, characterisation and modification of 1-deoxy-d-xylulose-5-phosphate synthase as a biocatalyst. Doctoral thesis , University of London.

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Abstract

The use of biotransformations in organic synthesis is a small but growing field. Biotransformations can be used for many steps which prove difficult with traditional chemistry, particularly stereospecific syntheses and chiral resolutions. However, to enable the increasing use of biotransformations in chemical synthesis, a broader range of biocatalysts will be required than those of which we are currently aware. Bioprospecting and genome sequencing are increasing the awareness of Nature's library of biocatalysts. These enzymes need to be characterised for their use as biocatalysts, and where necessary modified to meet needs that may not be catered for in nature. One currently used biocatalyst is transketolase. Transketolase is an enzyme which is produced by most organisms. As a biocatalyst it is most often extracted from spinach, or produced by recombinant Escherichia coli. Transketolase carries out a stereospecific carbon-carbon bond formation, removing a 2-carbon ketol group from a ketose sugar, and adding it to the aldehyde group of the aldose sugar. It is a useful biocatalyst as it has a broad substrate specificity. Significantly it will use hydroxypyruvate as the ketol donor, thus liberating carbon dioxide and driving the reaction in a forward direction. In this thesis the transketolase-like enzyme, 1-Deoxy-D-xylulose 5-Phosphate Synthase (DOX-P Synthase), is identified as another possible biocatalyst. Significantly DOX-P Synthase catalyses the addition of a carbonyl unit, not from hydroxypyruvate, but from pyruvate, a reaction which cannot be catalysed by transketolase. This thesis therefore describes studies that aimed to produce recombinant DOX-P Synthase as a biocatalyst, and to optimise its production. Further studies aimed to characterise the properties of DOX-P Synthase, with the aim of optimising its use as a biocatalyst. Analysis of the substrate range of DOX-P Synthase could then be used to describe the set of reactions for which it would be a useful biocatalyst. Finally, this thesis describes modifications made to the enzyme, carried out by site-directed mutagenesis, and the effects on enzyme properties.

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