Using Mutability Landscapes To Guide Enzyme Thermostabilization

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 4oxalocrotonate tautomerase (4-OT), which has potential as a biocatalyst for pharmaceutical synthesis, that gave rise to significant increases in apparent melting temperature T m (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 T m value, with no reduction in catalytic activity or enantioselectivity. This study illustrates the power of mutability-landscape-guided protein engineering for thermostabilizing enzymes.

Enantiocomplementary Michael Additions of Acetaldehyde to Aliphatic Nitroalkenes Catalyzed by Proline‐Based Carboligases

“…Using previously developed chemoenzymatic cascade reactions, the prepared γ‐nitroaldehydes can be readily converted in two steps into the respective GABA analogous. [16] Together with our continued protein engineering efforts to improve 4‐OT's stability and activity,[ 26 , 27 ] this work further highlights the potential of proline‐based carboligases as powerful catalysts for abiological carbon‐carbon bond‐forming reactions.…”

Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization

“…Over the past two decades, several protein engineering strategies have been investigated for enhancing the thermostability of enzymes, including directed evolution using random, 15 , 16 structure-guided, 17 , 18 or global mutagenesis 19 as well as bioinformatic approaches based on consensus mutagenesis 9 , 20 − 23 and ancestral sequence reconstruction. 24 − 26 Rational design strategies have included the introduction of intramolecular disulfide bridges, 27 − 30 targeted mutagenesis to flexible regions within the protein, 31 , 32 and computational design methods.…”

“…Over the past two decades, several protein engineering strategies have been investigated for enhancing the thermostability of enzymes, including directed evolution using random, , structure-guided, , or global mutagenesis as well as bioinformatic approaches based on consensus mutagenesis ,− and ancestral sequence reconstruction. − Rational design strategies have included the introduction of intramolecular disulfide bridges, − targeted mutagenesis to flexible regions within the protein, , and computational design methods. − Despite this progress, identifying beneficial mutations that stabilize an enzyme scaffold without sacrificing catalytic activity and/or without the requirement of extensive screening efforts has remained a considerable challenge. Protein stabilization by means of genetically encoded amino acids or chemical cross-linkers was also investigated. ,− …”