Anna Fryszkowska

Biocatalysis with thermostable enzymes: Structure and properties of a thermophilic “ene”-reductase related to Old Yellow Enzyme

We report the crystal structure of a thermophilic “ene” reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (Tm>70u2009°C) and shows increased resistance to denaturation in water‐miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene‐reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water‐miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity.

We have conducted a site‐specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,β‐unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2‐phenyl‐1‐nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to Cuf8feC reduction. These mutants showed an enhancement in a minor side reaction and formed 2‐phenylpropanal oxime from 2‐phenyl‐1‐nitropropene. The multiple binding conformations of hydroxy substituted nitro‐olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.

Focused Directed Evolution of Pentaerythritol Tetranitrate Reductase by Using Automated Anaerobic Kinetic Screening of Site-Saturated Libraries

This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96‐well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site‐saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)‐2‐aryl‐1‐nitropropene and α‐methyl‐trans‐cinnamaldehyde, respectively, with an increase in ee (62u2009% (R) for W102F). In addition, the detection of mutants with weak activity against α,β‐unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.

Active site modifications in pentaerythritol tetranitrate reductase can lead to improved product enantiopurity, decreased by-product formation and altered stereochemical outcome in reactions with α,β-unsaturated nitroolefins

This work describes a site-directed mutagenesis study of pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of key active site residues in influencing both product enantiopurity and the ratio of CC vs. nitro-group reduction with 2-phenyl-1-nitropropene. Comparative biotransformations of wild type and single/double mutants of PETN reductase with 2-phenyl-1-nitropropene showed that one enzyme scaffold was capable of generating both enantiomeric products with improved enantiopurities by a manipulation of the reaction conditions and/or the presence of a one or two key mutations. These changes located at key active site residues were sufficient to moderately improve product enantiopurity, cause a switch in the major product enantiomer formed and/or promote or eliminate side-product formation. The mutation of substrate-binding residue Y351 to alanine and phenylalanine improved the biocatalytic potential of PETN reductase by the elimination of a competing side reaction. The crystal structures of three mutants at residue Y351 (PDB codes: 3P81, 3P84 and 3P8J) show that only subtle changes in the active site environment may be necessary to generate significantly improved biocatalysts.

A short, chemoenzymatic route to chiral β-aryl-γ-amino acids using reductases from anaerobic bacteria

A short chemoenzymatic synthesis of beta-aryl-gamma-aminobutyric acids has been developed, based on a highly enantioselective biocatalytic reduction of beta-aryl-beta-cyano-alpha,beta-unsaturated carboxylic acids.

A surprising observation that oxygen can affect the product enantiopurity of an enzyme‐catalysed reaction

Enzymes are natural catalysts, controlling reactions with typically high stereospecificity and enantiospecificity in substrate selection and/or product formation. This makes them useful in the synthesis of industrially relevant compounds, particularly where highly enantiopure products are required. The flavoprotein pentaerythritol tetranitrate (PETN) reductase is a member of the Old Yellow Enzyme family, and catalyses the asymmetric reduction of β‐alkyl‐β‐arylnitroalkenes. Under aerobic conditions, it additionally undergoes futile cycles of NAD(P)H reduction of flavin, followed by reoxidation by oxygen, which generates the reactive oxygen species (ROS) hydrogen peroxide and superoxide. Prior studies have shown that not all reactions catalysed by PETN reductase yield enantiopure products, such as the reduction of (E)‐2‐phenyl‐1‐nitroprop‐1‐ene (PNE) to produce (S)‐2‐phenyl‐1‐nitropropane (PNA) with variable enantiomeric excess (ee). Recent independent studies of (E)‐PNE reduction by PETN reductase showed that the major product formed could be switched to (R)‐PNA, depending on the reaction conditions. We investigated this phenomenon, and found that the presence of oxygen and ROS influenced the overall product enantiopurity. Anaerobic reactions produced consistently higher nitroalkane (S)‐PNA product yields than aerobic reactions (64% versus 28%). The presence of oxygen dramatically increased the preference for (R)‐PNA formation (up to 52% ee). Conversely, the presence of the ROS superoxide and hydrogen peroxide switched the preference to (S)‐PNA product formation. Given that oxygen has no role in the natural catalytic cycle, these findings demonstrate a remarkable ability to manipulate product enantiopurity of this enzyme‐catalysed reaction by simple manipulation of reaction conditions. Potential mechanisms of this unusual behaviour are discussed.