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.
Submitter: Shu-Ching Ou
Submission Date: March 28, 2019, 10 a.m.
|Number of data points||70|
|Assays/Quantities/Protocols||Experimental Assay: Specific Activity at 22 °C ; Experimental Assay: Specific Activity at 60 °C ; Experimental Assay: Tm ; Experimental Assay: t1/2 ; Experimental Assay: Km ; Experimental Assay: kcat ; Experimental Assay: kcat/Km|
|Libraries||Variants for TK|
|Structure ID||Release Date||Resolution||Structure Title|
|6TJ8||2020-07-08||0.92||Escherichia coli transketolase in complex with cofactor analog 2'-methoxythiamine diphosphate|
|6TJ9||2020-07-08||0.95||Escherichia coli transketolase in complex with cofactor analog 2'-methoxythiamine and substrate xylulose 5-phosphate|
|6RJC||2019-09-11||1.05||E.coli transketolase apoenzyme|
|2R8O||2007-12-11||1.47||Transketolase from E. coli in complex with substrate D-xylulose-5-phosphate|
|5HHT||2016-11-02||1.5||Crystal structure of E. coli transketolase triple variant Ser385Tyr/Asp469Thr/Arg520Gln|
|2R5N||2007-11-06||1.6||Crystal structure of transketolase from Escherichia coli in noncovalent complex with acceptor aldose ribose 5-phosphate|
|2R8P||2007-12-11||1.65||Transketolase from E. coli in complex with substrate D-fructose-6-phosphate|
|1QGD||2003-06-17||1.9||TRANSKETOLASE FROM ESCHERICHIA COLI|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|