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 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.

Submission Details

ID: SCKNfGn

Submitter: Shu-Ching Ou

Submission Date: March 28, 2019, 10 a.m.

Version: 1

Publication Details
Yu H;Yan Y;Zhang C;Dalby PA,Sci Rep (2017) Two strategies to engineer flexible loops for improved enzyme thermostability. PMID:28145457
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
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

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
199.6 A,B Transketolase 1 P27302 TKT1_ECOLI