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

• Published in: The FEBS journal 2012-11, Vol.279 (22), p.4160-4171
• Main Authors: Fryszkowska, Anna, Toogood, Helen S., Mansell, David, Stephens, Gill, Gardiner, John M., Scrutton, Nigel S.
• Format: Article
• Language: English
• Subjects: biotransformations; Catalysis; Chemical reactions; Cycloparaffins - chemistry; Cycloparaffins - metabolism; Cysteine - metabolism; Enzymes; Hydrogen Peroxide - metabolism; Hydrogen-Ion Concentration; Kinetics; Methionine - metabolism; NADP - metabolism; nitroolefin reduction; Nitroparaffins - metabolism; Oxidoreductases - metabolism; Oxygen; Oxygen - metabolism; pentaerythritol tetranitrate reductase; Propane - analogs & derivatives; Propane - metabolism; Proteins; reactive oxygen species; Reactive Oxygen Species - metabolism; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism; Superoxides - metabolism
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12008

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. Pentaerythritol tetranitrate reductase catalyses the NAD(P)H‐dependent reduction of (E)‐2‐phenyl‐1‐nitroprop‐2‐ene. Under aerobic conditions, it undergoes FMNred reoxidation by oxygen, generating hydrogen peroxide and superoxide (ROS). The presence of oxygen and ROS influenced 2‐phenyl‐1‐nitropropane product enantiopurity, by increasing the proportion of (R)‐ and (S)‐alkane generated, respectively. This may be due to the presence of oxidatively modified cysteine and/or methionine residues.