Abstract
Monoterpenoids are the main components of plant essential oils and the active components of some traditional Chinese medicinal herbs like Mentha haplocalyx Briq., Nepeta tenuifolia Briq., Perilla frutescens (L.) Britt and Pogostemin cablin (Blanco) Benth. Pulegone reductase is the key enzyme in the biosynthesis of menthol and is required for the stereoselective reduction of the Δ2,8 double bond of pulegone to produce the major intermediate menthone, thus determining the stereochemistry of menthol. However, the structural basis and mechanism underlying the stereoselectivity of pulegone reductase remain poorly understood. In this study, we characterized a novel (−)-pulegone reductase from Nepeta tenuifolia (NtPR), which can catalyze (−)-pulegone to (+)-menthone and (−)-isomenthone through our RNA-seq, bioinformatic analysis in combination with in vitro enzyme activity assay, and determined the structure of (+)-pulegone reductase from M. piperita (MpPR) by using X-ray crystallography, molecular modeling and docking, site-directed mutagenesis, molecular dynamics simulations, and biochemical analysis. We identified and validated the critical residues in the crystal structure of MpPR involved in the binding of the substrate pulegone. We also further identified that residues Leu56, Val282, and Val284 determine the stereoselectivity of the substrate pulegone, and mainly contributes to the product stereoselectivity. This work not only provides a starting point for the understanding of stereoselectivity of pulegone reductases, but also offers a basis for the engineering of menthone/menthol biosynthetic enzymes to achieve high-titer, industrial-scale production of enantiomerically pure products.
Highlights
Terpenoids are a large and structurally diverse group of natural products widely distributed in plants, microorganisms, and insects
The C10 menthol skeleton is formed by the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) (Gao et al, 2020), which are obtained from the 2C-methyl-D-erythritol4-phosphate (MEP) pathway or the mevalonic acid (MVA) pathway in plant cells, followed by cyclization catalyzed by (−)-limonene synthase (LS)
Pulegone reductase from Mentha piperita (MpPR) that catalyzes the reduction of the C2–C8 double bond of (+)-pulegone to (−)menthone using NADP(H) as a co-factor has been established for decades (Lange, 2015)
Summary
Terpenoids are a large and structurally diverse group of natural products widely distributed in plants, microorganisms, and insects. More than 55,000 terpenoids have been identified so far, and the majority of them display diverse biological activities (Christianson, 2008). Monoterpenoids are a type of terpenoids composed of two isoprene units and are widely distributed in plants with great therapeutic potential, such as traditional Chinese medicinal herbs like Mentha haplocalyx Briq (Duan et al, 2015), Nepeta tenuifolia Briq (Liu et al, 2018), Perilla frutescens (L.) Britt (Zhou et al, 2021), and Pogostemin cablin (Blanco) Benth (Wojtunik-Kulesza et al, 2019). As one of the representative monoterpenoids, menthol and its biosynthetic pathway as well as related biosynthetic enzymes have been identified (Gao et al, 2020). The C10 skeleton is further modified by a series of tailoring enzymes (e.g., monooxygenase, dehydrogenase, reductase, and isomerase) to generate (−)-menthol and the diastereomer (+)-isomenthol (Supplementary Figure 1A)
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