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

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.

Deciphering the Unconventional Reduction of C═N Bonds by Old Yellow Enzymes Using QM/MM

“…From here, O1 could fetch a proton from nearby water molecules or Y27; we have considered both possibilities and found that proton transfer from the water has a lower energy barrier (Figure S8). This is consistent with our previous experimental observation that the replacement of Y27 by phenylalanine did not significantly impact the overall reaction . Although the optimized molecular structures corresponding to stationary points along the lowest energy reaction coordinate for the direct proton transfer from Y27 are shown in (Figure S9), here we are considering water as a proton donor for the general case.…”

“…Due to the similarity of the chemical skeleton of imine 2 and oxime 1 , it is very likely that the imine 2 binds the same way as the oxime 1 in normalR normalS normalH − 1 . Also, our previous MD simulations indicated that both 1 and 2 have similar binding poses . Additionally, the docked pose of 2 is also very similar to our modeled complex of 2 in the active site of XenA, which has been modeled by using the binding pose of 1 as a template.…”

“…The calculated activation energy (Δ E a ‡ ) for the hydride transfer is 14.19 kcal/mol, whereas the computed free energy (Δ G ‡ ) change for the same is 9.82 kcal/mol (Figure S4). In our previous work, the presteady-state rate for the reoxidation of flavin by the substrate 1 ( k ox ) was estimated to be 28.7 ± 3.2 s –1 . Applying classical transition-state theory using the Eyring equation, this corresponds to a Δ E a ‡ of 15.4 kcal/mol.…”

Enzyme‐Triggered Reactions for the Synthesis of Organic Molecules

“…[15] The MAOÀ N D9 variant was used to convert pyrrolidine 36 to the corresponding pyrroline 37, which triggered a spontaneous intramolecular oxidative Pictet-Spengler cyclisation to afford compound 38. The natural product (R)harmicine (35) could then be obtained from racemic 38 in the chemoenzymatic non-selective reduction/selective oxidation approach, using ammonium borohydride along with the MAOÀ N D9. [5] Cyclic electrophilic imines have also been produced by ATAcatalysed conversion of diamines 39 to aminoaldehyde intermediates 40, which instantaneously convert to their cyclisation products 41.…”

“…[34] This enzyme-catalysed reaction triggers a subsequent spontaneous dimerisation and oxidation of the amine 94 to form the pyrazine product 95. While the focus of this work by Velikogne and Breukelaar et al was to uncover the mechanism of the ene-reductase-catalysed reaction, [35] the pyrazine 95 formation from an oxime substrate 93 is another interesting enzyme-triggered reaction with the spontaneous aromatisation of the amine 94 acting as the driving force for the reaction.…”