Pinpointing a Mechanistic Switch Between Ketoreduction and “Ene” Reduction in Short‐Chain Dehydrogenases/Reductases

Abstract

Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (−)‐menthone:(−)‐menthol reductase and (−)‐menthone:(+)‐neomenthol reductase, and the “ene” reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue‐swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β‐unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases.

Crossing the Border: From Keto‐ to Imine Reduction in Short‐Chain Dehydrogenases/Reductases

“…With the latter activity being engineered, our work has revealed that the catalytic scope of SDR enzymes can be manipulated, which is in line with the results by Lygidakis et al. concerning C=O and enone reduction . Moreover, our results show that the starting activity is not a prerequisite for obtaining a desired novel functionality, here imine reduction.…”

“…Notably, Lygidakis et al. used protein engineering to address the catalytic scope of SDRs: the exchange of a single residue enabled the switch from C=C (enone) to C=O reduction in SDRs from Mentha piperita …”

Discovery of a Short‐Chain Dehydrogenase from Catharanthus roseus that Produces a New Monoterpene Indole Alkaloid

“…To understand the mechanism of VAS better, the crystal structure was solved (Table and Figure ; PDB ID: 5O98). It closely resembles the structure of salutaridine reductase from Papaver somniferum (SalR; PDB ID: 3O26; i.e., the molecular replacement template) and the recently determined structures of two SDRs from Mentha piperita : menthone‐neomenthol reductase (MNMR; e.g., PDB ID: 5L53) and isopiperitenone reductase (IPR; e.g., PDB ID: 5LCX; Figure S10). Crystallization of VAS was carried out with and without the oxidized cofactor NADP + .…”

Discovery, Characterization, Engineering, and Applications of Ene-Reductases for Industrial Biocatalysis

“…The ER isopiperitenone reductase (IPR) catalyses the C=C reduction of isopiperitenone to cis -isopulegone, while ketoreductases (—)-menthone:(—)-menthol reductase (MMR) and (—)-menthone:(+)-neomenthol reductase (MNMR) reduce menthone and isomenthone to menthol isomers (Scheme 11). 20 The identification of a catalytic residue substitution (IPR E238 to MNMR Y244) followed by residue-swapping mutagenesis and X-ray structure determination suggested this change was a likely cause of the switch from ene-reduction to ketoreduction, respectively.…”