Abstract
A novel transketolase has been reconstituted from two separate polypeptide chains encoded by a ‘split-gene’ identified in the genome of the hyperthermophilic bacterium, Carboxydothermus hydrogenoformans. The reconstituted active α2β2 tetrameric enzyme has been biochemically characterized and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to near 100% completion due to the release of the product carbon dioxide and can be used for the synthesis of a range of sugars of interest to the pharmaceutical industry. This novel reconstituted transketolase is thermally stable with no loss of activity after incubation for 1 h at 70°C and is stable after 1 h incubation with 50% of the organic solvents methanol, ethanol, isopropanol, DMSO, acetonitrile and acetone. The X-ray structure of the holo reconstituted α2β2 tetrameric transketolase has been determined to 1.4 Å resolution. In addition, the structure of an inactive tetrameric β4 protein has been determined to 1.9 Å resolution. The structure of the active reconstituted α2β2 enzyme has been compared to the structures of related enzymes; the E1 component of the pyruvate dehydrogenase complex and D-xylulose-5-phosphate synthase, in an attempt to rationalize differences in structure and substrate specificity between these enzymes. This is the first example of a reconstituted ‘split-gene’ transketolase to be biochemically and structurally characterized allowing its potential for industrial biocatalysis to be evaluated.
Highlights
Transketolase (TK, EC 2.2.1.1) is a thiamine diphosphate-dependant (TPP) enzyme which plays an important role in the pentose phosphate pathway
The ChTK-N and ChTK-C genes were successfully cloned into the pLATE51 (Thermo Scientific) expression vector and over expressed in E. coli BL21 DE3∗ using the auto induction ZYM5052 medium (Studier, 2005)
The formation of the complex was analyzed using size exclusion chromatography (GF-200) (Figure 1) in which a slight decrease in MW can be observed between the ChTK-F and ChTK-C peaks as well as small ChTK-N peak corresponding to the excess ChTK-N used in the reaction
Summary
Transketolase (TK, EC 2.2.1.1) is a thiamine diphosphate-dependant (TPP) enzyme which plays an important role in the pentose phosphate pathway It catalyses the rearrangement of sugar molecules by the transfer of a C2 unit from D-xylulose-5-phosphate to erythrose-4-phosphate, resulting in the formation of fructose-6-phosphate and glyceraldehyde-3-phosphate which are fed back into the glycolysis pathway (Racker et al, 1954). The ability of TKs to form enantioselective carbon-carbon bonds has generated increasing interest for their use as biocatalysts in industrial synthetic reactions (Hibbert et al, 2008) This reaction has previously been described as irreversible due to the production and evolution of CO2 but was shown by Marsden et al (2017) to be reversible over a period of weeks because while the decarboxylation of hydroxypyruvate is virtually irreversible, the carbon-carbon bond formation is not. The Escherichia coli TK (EcTK) has previously been used to demonstrate the potential synthesis of a range of pharmaceutical relevant sugars with HPA as the ketol donor and glycolaldehyde as the acceptor to produce the sugar erythrulose at a 100% conversion (Lilly et al, 1996)
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