Using Mutability Landscapes To Guide Enzyme Thermostabilization

Chao Guo, Yan Ni, Lieuwe Biewenga, Tjaard Pijning, Andy-Mark W. H. Thunnissen, Gerrit J. Poelarends*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

46 Downloads (Pure)

Abstract

Thermostabilizing enzymes while retaining their activity and enantioselectivity for applied biocatalysis is an important topic in protein engineering. Rational and computational design strategies as well as directed evolution have been used successfully to thermostabilize enzymes. Herein, we describe an alternative mutability-landscape approach that identified three single mutations (R11Y, R11I and A33D) within the enzyme 4-oxalocrotonate tautomerase (4-OT), which has potential as a biocatalyst for pharmaceutical synthesis, that gave rise to significant increases in apparent melting temperature Tm (up to 20 °C) and in half-life at 80 °C (up to 111-fold). Introduction of these beneficial mutations in an enantioselective but thermolabile 4-OT variant (M45Y/F50A) afforded improved triple-mutant enzyme variants showing an up to 39 °C increase in Tm value, with no reduction in catalytic activity or enantioselectivity. This study illustrates the power of mutability-landscape-guided protein engineering for thermostabilizing enzymes.

Original languageEnglish
Pages (from-to)170-175
Number of pages7
JournalChemBioChem
Volume22
Issue number1
Early online date30-Sep-2020
DOIs
Publication statusPublished - 5-Jan-2021

Keywords

  • biocatalysis
  • mutability landscape
  • protein engineering
  • thermostability
  • DIRECTED EVOLUTION
  • STABILITY
  • TAUTOMERASE
  • ALDOLASE

Cite this