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.

Keywords: Mentha essential oil biosynthesis; biotransformations; isopiperitenone reductase; short-chain dehydrogenases/reductases; structure elucidation.

Scheme 1

Reactions catalyzed by MMR, MNMR, and IPR.1a,1b

Figure 1

Crystal structure analyses of IPR and MNMR. A) Overlay of IPR (blue; PDB code 5LCX) and SalR (coral; PDB code 3O26) structures. The flap domains of IPR and SalR are indicated by dotted lines. NADP⁺ is displayed as ball and stick and colored by atom type. B) Left: overlay of IPR (gray; PDB code 5LDG) and MNMR (yellow; PDB code 5L53) structures. Right: active site showing side chains of some active‐site residues of IPR and MNMR along with 3 a (cyan) and NADP⁺. The Figure was prepared using CCP4mg.11

Scheme 2

Proposed mechanisms of A) ketoreduction by MNMR and B) reduction of an α,β‐unsaturated double bond by IPR. The three‐dimensional nature of the active sites is represented as compounds in the foreground and background shown in black and grey, respectively.