title: 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 kcatand a Tmincreased 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.
authors.affiliation: Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, United Kingdom.
authors.forename: Haoran
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, United Kingdom.
authors.forename: Yihan
authors.initials: Y
authors.lastname: Yan
authors.affiliation: Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, United Kingdom.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Department of Biochemical Engineering, University College London, Gordon Street, London, WC1H 0AH, United Kingdom.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Dalby
mesh_terms: D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D006358:Hot Temperature; D006860:Hydrogen Bonding; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D013816:Thermodynamics; D014174:Transketolase / Q000737:chemistry* / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; D014174:Transketolase