Abstract
Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of Escherichia coli transketolase (TK). The first was a “back to consensus mutations” approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes.
Highlights
Transketolase (TK), a thiamine diphosphate dependent (ThDP) enzyme, catalyses the reversible transfer of a C2-ketol unit from D-xylulose-5-phosphate to either D-ribose-5-phosphate or D-erythrose-4-phosphate, linking glycolysis to the pentose phosphate pathway in all living cells[1,2]
It remains a challenge to design efficient bioconversions of aliphatic or aromatic aldehyde substrates by E. coli transketolase, at elevated temperatures to enhance their solubility in water
Our recent mutagenesis of cofactor-binding loops towards those amino-acids found in Thermus thermophilus at equivalent positions, provided some success in which the H192P variant increased the optimal temperature for activity from 55 °C to 60 °C, with a linked increase in the Tagg measured by dynamic light scattering, from 60 °C to Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, United Kingdom
Summary
Transketolase (TK), a thiamine diphosphate dependent (ThDP) enzyme, catalyses the reversible transfer of a C2-ketol unit from D-xylulose-5-phosphate to either D-ribose-5-phosphate or D-erythrose-4-phosphate, linking glycolysis to the pentose phosphate pathway in all living cells[1,2]. The use of β-hydroxypyruvate (HPA) as the ketol donor renders the donor-half reaction irreversible, increasing the atom efficiency of the reaction favourably for industrial syntheses. As a mesophilic enzyme E. coli TK suffers the limitation of low stability to elevated temperatures and extremes of pH14, limiting its current use in industrial processes. It remains a challenge to design efficient bioconversions of aliphatic or aromatic aldehyde substrates by E. coli transketolase, at elevated temperatures to enhance their solubility in water. Our recent mutagenesis of cofactor-binding loops towards those amino-acids found in Thermus thermophilus at equivalent positions, provided some success in which the H192P variant increased the optimal temperature for activity from 55 °C to 60 °C, with a linked increase in the Tagg measured by dynamic light scattering, from 60 °C to www.nature.com/scientificreports/. Considerable improvement is still required in both the specific activity and the half-life of the enzyme at elevated temperatures, to achieve good conversion yields
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