Tuning Enzyme Activity for Nonaqueous Solvents: Engineering an Enantioselective “Michaelase” for Catalysis in High Concentrations of Ethanol

Abstract

Enzymes have evolved to function under aqueous conditions and may not exhibit features essential for biocatalytic application, such as the ability to function in high concentrations of an organic solvent. Consequently, protein engineering is often required to tune an enzyme for catalysis in non‐aqueous solvents. In this study, we have used a collection of nearly all single mutants of 4‐oxalocrotonate tautomerase, which promiscuously catalyzes synthetically useful Michael‐type additions of acetaldehyde to various nitroolefins, to investigate the effect of each mutation on the ability of this enzyme to retain its “Michaelase” activity in elevated concentrations of ethanol. Examination of this mutability landscape allowed the identification of two hotspot positions, Ser30 and Ala33, at which mutations are beneficial for catalysis in high ethanol concentrations. The “hotspot” position Ala33 was then randomized in a highly enantioselective, but ethanol‐sensitive 4‐OT variant (L8F/M45Y/F50A) to generate an improved enzyme variant (L8F/A33I/M45Y/F50A) that showed great ethanol stability and efficiently catalyzes the enantioselective addition of acetaldehyde to nitrostyrene in 40 % ethanol (permitting high substrate loading) to give the desired γ‐nitroaldehyde product in excellent isolated yield (89 %) and enantiopurity (ee=98 %). The presented work demonstrates the power of mutability‐landscape‐guided enzyme engineering for efficient biocatalysis in non‐aqueous solvents.

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.…”

Using Mutability Landscapes To Guide Enzyme Thermostabilization

“…Analysis of the mutability landscape revealed two “hotspot” positions, Arg11 and Ala33, at which single mutations resulted in enzyme variants that showed more than 50 % remaining activity at the elevated temperature. Interestingly, mutant A33D, which has previously been shown to possess high enantioselectivity and increased “Michaelase” activity, [6a] as well as improved stability towards high ethanol concentrations, [8c] appears to also have enhanced thermostability.…”

“…The values shown indicate mean ± standard deviation from three parallel experiments. [b] Determination of absolute configuration was based on chiral GC and previously reported chiral GC data with authentic standards (Figure S1) [6a,8c] …”

“…Interestingly, residue Ala33 was previously identified as a “hotspot” position at which mutations are beneficial for catalysis in high concentrations of the cosolvent ethanol [8c] . Particularly, the 4‐OT variant A33D not only showed improved ethanol tolerance, [8c] but also enhanced Michael‐type addition activity and enantioselectivity [6a] . This single point mutation thus has a beneficial effect on the activity, stereoselectivity, solvent stability and thermostability of 4‐OT.…”

Exploring Oxidoreductases from Extremophiles for Biosynthesis in a Non-Aqueous System

“…Strengthening protein surface hydration via regulating the surface charge is an effective and efficient strategy for tailoring enzyme stability and improving their resistance in organic solvents [ 51 ]. It is found that buried waters contribute most to rapid equilibration in organic solvents, with slow-diffusing waters giving similar results [ 52 ]. Enzyme gates are key residues in the substrate tunnel that control reversible conformational changes.…”