Two strategies to engineer flexible loops for improved enzyme thermostability

Yu, Haoran, Yan, Yihan, Zhang, Cheng and Dalby, Paul A. 2017. Two strategies to engineer flexible loops for improved enzyme thermostability. Scientific Reports 7 (1) , 41212. 10.1038/srep41212

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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.

Item Type:	Article
Date Type:	Publication
Status:	Unpublished
Schools:	Biosciences
Publisher:	Nature Research
ISSN:	2045-2322
Date of Acceptance:	16 December 2016
Last Modified:	26 Nov 2024 11:16
URI:	https://orca.cardiff.ac.uk/id/eprint/173711

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