Two strategies to engineer flexible loops for improved enzyme thermostability

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 wildtype 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 k cat and a T m 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.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 stereospecifically controlled carbon-carbon bond forming ability of TK makes it promising as a biocatalyst in industry, for the synthesis of complex carbohydrates and other high-value compounds 3,4 . The use of β -hydroxypyruvate (HPA) as the ketol donor renders the donor-half reaction irreversible, thus increasing the atom efficiency of the reaction favourably for industrial syntheses. Escherichia coli (E. coli) TK converts HPA with a rate of 60 U/mg, significantly higher than the rates of 2 U/mg and 9 U/mg reported for its orthologs from spinach and yeast 5 .Wild-type (WT) E. coli TK has been successfully engineered to have improved and inverted enantioselectivity 6 , as well as an expanded aldol-acceptor substrate range including polar aliphatics 7 , non-polar aliphatics 8 , and heteroaromatics 9,10 . Most recently, E. coli TK has been engineered to synthesize L-gluco-heptulose from L-arabinose, thus transforming a major component of the carbohydrates in sugar beet pulp, into a rare naturally-occurring ketoheptose with potential therapeutic applications in hypoglycaemia and cancer 11 . Additionally, the substrate range accepted by TK has been recently extended to aromatic benzaldehyde derivatives, which opened up potential routes to chiral aromatic amino-alcohols such as chloramphenicol antibiotics, nor-ephedrine, and their analogues with alternative aromatic substituents 12,13 .As a mesophilic enzyme E. coli TK suffers the limitation of low stability t...

Biotransformation of β‐hydroxypyruvate and glycolaldehyde to l‐erythrulose by Pichia pastoris strain GS115 overexpressing native transketolase

“…Transitioning from harsh to mild conditions between process steps typically requires further energy or material inputs and brings significant cost implications . Purified enzymes are often active in only mild conditions, but variants can be sourced or engineered that are stable to extremes . However, even robust enzyme variants have no capacity to regenerate themselves, as they inevitably accumulate damage and denaturation over time.…”

“…5 Purified enzymes are often active in only mild conditions, but variants can be sourced or engineered that are stable to extremes. 6 However, even robust enzyme variants have no capacity to regenerate themselves, as they inevitably accumulate damage and denaturation over time. One option to make enzymes both selfrenewing and tolerant to non-mild conditions is to deploy them while they are still sequestered within a host cell, socalled whole cell biocatalysis.…”

Biophysical characterization of the inactivation ofE. colitransketolase by aqueous co-solvents

“…Finally, this work has provided useful insights that will guide the future engineering of TK and other similarly complex enzymes. Improvements might be obtained by focussing enzyme engineering within the cofactor-binding loops and neighbouring regions close to the protein surface, as we have shown previously to be effective for the improvement of TK thermostability (Yu et al, 2017). Table 3.…”

“…The synthetic potential of TK has already been significantly improved by successive rounds of directed evolution with smart libraries (; Affaticati et al, 2016;Yu et al 2017), to enhance activity towards polar Subrizi et al, 2016), and increasingly aliphatic Cazares et al, 2010: Strafford et al, 2012, and heterocyclic substrates (Cazares et al 2010;Galman et al, 2010), to enhance and reverse enantioselectivity . De novo pathways containing improved TK variants have also been designed for the synthesis of optically pure high-value amino-diols (Rios-Solis et al, 2011.…”

“…Additionally, effects of varying cofactor concentrations on TK activity in holo-TK and apo-TK have been observed, which the latter showed inactivity in the lysate wild-type TK (Miller et al, 2007). Recently, a mutation to proline in one of the two TK cofactor-binding loops was found to stabilise TK to aggregation at elevated temperatures (Morris et al, 2016); and based on this, flexible loops within the structure were engineered and found improvement in thermostability (Yu et al 2017). Thus, one or more of several potential mechanisms could influence TK stability and activity in solvents, including: i) global unfolding; ii) aggregation; iii) partial unfolding of local structure or domains; iv) cofactor dissociation; and v) modulating the accessibility of water molecules or substrates into the active site.…”