Characterisation of the structural stability of transketolase under biocatalytically relevant conditions

Abstract

The enzyme transketolase (TK; E.C. 2.2.1.1) from Escherichia coli occupies a pivotal place in metabolic regulation. TK catalyses the interconversion of sugars by transferring a two-carbon ketol unit from a ketose donor substrate to an aldolase acceptor substrate. It is also an important biocatalyst in stereo-specific carbon-carbon bond synthesis with potential industrial application for the synthesis of pharmaceuticals, agrochemicals and fine chemicals. Although many useful reactions have been reported for TK, many of the substrates and products are unstable or insoluble at the pH or temperature for which the enzyme has optimum activity. Understanding the structural stability of transketolase under bioprocess conditions will improve our capacity to comprehend and ultimately to engineer it to make it work in a broader range of pH or temperature to potentially help in the reduction of process time and to increase the quality and solubility of products. In this research I characterised the early events on the urea denaturation pathway of E. coli transketolase, providing new insights into the mechanisms of enzyme deactivation that occur under biocatalytic conditions. Equilibrium denaturation measurements by fluorescence intensity and circular dichroism (CD), combined with size-exclusion and dynamic light scattering studies, have revealed three transitions in the denaturation pathway for holo-TK. The first step, at low urea concentration corresponds to the local restructuring of the thiamine diphosphate (TPP) binding-sites. Next, the dissociation of the TPP cofactors and partial loss of secondary structure produces a form, which is most consistent with a partially denatured dimeric enzyme. While the enzyme is deactivated initially by changes in structure associated with the cofactors, this event does not release the cofactor from the enzyme, consistent with the intermediate formed during the reconstitution of holo-TK from apo-TK. Improvement of biocatalytic processes using TK over prolonged reaction times would, therefore, need to address the formation of this cofactor-associated intermediate state. Equivalent results were also observed with a high throughput microplate-based fluorescence method that uses less enzyme and time. The equilibrium denaturation of holo and apo-TK at different temperatures and pH was also investigated for further insights into the enzyme stability and to provide a benchmark for assessing any future enzyme variants with altered pH or temperature optima. In an effort to enhance the stability of the enzyme I subsequently used bioinformatical, statistical and multivariate analyses of protein sequences and associated properties to determine the most likely residues to affect temperature and pH optima in biocatalysis. The outcome of this first parametric statistical analysis (Pearson’s r test) rendered 20 different points. Promising mutation points were selected based on the correlation coefficient (r) results taken into account a level of significance α = 0.05. TK E. coli selected points were then mutated and screened by site-saturated mutagenesis (SSM) and automated techniques respectively. Finally alternative statistical correlation methods were examined, including a non-parametric statistical analysis (Kendall’s τ or tau test), principal component analysis (PCA) and partial least square (PLS), for their potential to generate TK mutant variants with either enhanced pH or temperature stability