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. 2023 Feb 6;13(4):2610-2618.
doi: 10.1021/acscatal.2c06137. eCollection 2023 Feb 17.

Mechanistic Insights into the Ene-Reductase-Catalyzed Promiscuous Reduction of Oximes to Amines

Willem B Breukelaar  1 , Nakia Polidori  2 , Amit Singh  2 , Bastian Daniel  2 , Silvia M Glueck  1 , Karl Gruber  2   3   4 , Wolfgang Kroutil  1   3   4
Affiliations

Mechanistic Insights into the Ene-Reductase-Catalyzed Promiscuous Reduction of Oximes to Amines

Willem B Breukelaar et al. ACS Catal. .

Abstract

The biocatalytic reduction of the oxime moiety to the corresponding amine group has only recently been found to be a promiscuous activity of ene-reductases transforming α-oximo β-keto esters. However, the reaction pathway of this two-step reduction remained elusive. By studying the crystal structures of enzyme oxime complexes, analyzing molecular dynamics simulations, and investigating biocatalytic cascades and possible intermediates, we obtained evidence that the reaction proceeds via an imine intermediate and not via the hydroxylamine intermediate. The imine is reduced further by the ene-reductase to the amine product. Remarkably, a non-canonical tyrosine residue was found to contribute to the catalytic activity of the ene-reductase OPR3, protonating the hydroxyl group of the oxime in the first reduction step.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1. Ene-Reductase-Catalyzed Transformations of α-Oximo β-Keto Esters
Scheme 2
Scheme 2. Possible Intermediates in the Reduction of Oxime 1 to Amine 2
Scheme 3
Scheme 3. Two-Step Reduction of Oximes 1b and 1c to Get Indications for the Formation of the Amino Moiety
The enzymes originate from L. esculentum (OPR3), P. putida (XenA), Rhodococcus ruber (ADH-A), and a variant of the ADH from L. kefir (LkADH-Lica). Reaction conditions: ene-reductase (4.89 μM for XenA and 4.46 μM for OPR3), 10 mM substrate, 0.5 mM NADPH, 0.5 mM reduced nicotinamide adenine dinucleotide (NADH) (only for reactions with ADH-A), 50 mM glucose, 4 mg/mL GDH, 5% dimethyl sulfoxide (DMSO) (v/v), 50 mM phosphate buffer, pH 7.5, 30 °C, 24 h, and 120 rpm. Total volume: 0.5 mL. Product formation is defined by the amount of substrate transformed to the amino alcohol (as the N-benzoyl derivative) as deduced from high-performance liquid chromatography (HPLC) analysis on a chiral stationary phase using calibration curves.
Figure 1
Figure 1
Crystal structures of OPR3/XenA in complex with substrates 1a and 1b. (a) OPR3/1a. (b) XenA/1a. (c) OPR3/1b. (d) XenA/1b.
Figure 2
Figure 2
Binding of oxime 1b in the active site of XenA obtained from MD simulation.
Figure 3
Figure 3
Pocket in the active site of XenA in the surface mode, harboring the propanoyl moiety of oxime 1b (top) and the active site cavity calculated using the HOLLOW program (bottom). The pocket bearing the propanoyl moiety is highlighted in green for clarity.
Scheme 4
Scheme 4. Possible Pathways Involving the Canonical and Non-canonical Tyrosine in Oxime Reduction
Figure 4
Figure 4
Stopped-flow pre-steady-state kinetics. Reductive (left) and oxidative (right) half-reaction of OPR3 (top)/XenA (bottom) and their variants.
Scheme 5
Scheme 5. Synthesis of Hydroxylamine 8
Scheme 6
Scheme 6. Pyrazine Formation of Oxime Methyl Ethers Catalyzed by EREDs
Scheme 7
Scheme 7. Unsuccessful Biotransformations of O-Methyl Hydroxylamine 8 to Pyrazine 2
Reaction conditions: ene-reductase (200 μg/mL, 4 μM), 10 mM substrate, 0.5 mM NADPH, 50 mM glucose, 4 mg/mL GDH, 5% DMSO (v/v), 50 mM phosphate buffer, pH 7.5, 30 °C, 24 h, and 120 rpm. Total volume: 0.5 mL.
Scheme 8
Scheme 8. Summary of the Mechanism of ERED-Catalyzed Reduction of α-Oximo-β-keto Esters
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