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Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed

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  • Dylan Alexander Carlin
  • Siena Hapig-Ward
  • Bill Wayne Chan
  • Natalie Damrau
  • Mary Riley
  • Ryan W Caster
  • Bowen Bethards
  • Justin B Siegel

Abstract

Accurate modeling of enzyme activity and stability is an important goal of the protein engineering community. However, studies seeking to evaluate current progress are limited by small data sets of quantitative kinetic constants and thermal stability measurements. Here, we report quantitative measurements of soluble protein expression in E. coli, thermal stability, and Michaelis-Menten constants (kcat, KM, and kcat/KM) for 129 designed mutants of a glycoside hydrolase. Statistical analyses reveal that functional Tm is independent of kcat, KM, and kcat/KM in this system, illustrating that an individual mutation can modulate these functional parameters independently. In addition, this data set is used to evaluate computational predictions of protein stability using the established Rosetta and FoldX algorithms. Predictions for both are found to correlate only weakly with experimental measurements, suggesting improvements are needed in the underlying algorithms.

Suggested Citation

  • Dylan Alexander Carlin & Siena Hapig-Ward & Bill Wayne Chan & Natalie Damrau & Mary Riley & Ryan W Caster & Bowen Bethards & Justin B Siegel, 2017. "Thermal stability and kinetic constants for 129 variants of a family 1 glycoside hydrolase reveal that enzyme activity and stability can be separately designed," PLOS ONE, Public Library of Science, vol. 12(5), pages 1-13, May.
    • Handle: RePEc:plo:pone00:0176255
    DOI: 10.1371/journal.pone.0176255
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    1. Jan-Ytzen van der Meer & Harshwardhan Poddar & Bert-Jan Baas & Yufeng Miao & Mehran Rahimi & Andreas Kunzendorf & Ronald van Merkerk & Pieter G. Tepper & Edzard M. Geertsema & Andy-Mark W. H. Thunniss, 2016. "Using mutability landscapes of a promiscuous tautomerase to guide the engineering of enantioselective Michaelases," Nature Communications, Nature, vol. 7(1), pages 1-16, April.
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