Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis

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.

DFT Studies of the Activity and Reactivity of Limonene in Comparison with Selected Monoterpenes

“…In the case of BDE calculated for cymene (Table S3, Scheme S1), the C8-H bond is the easiest to break, which is also confirmed by the products obtained in preparative experiments [92]. In thymol and menthol molecules, the breaking of bonds leading to the formation of the corresponding ketones (Tables S5 and S6, Scheme S1) is favored [77,[93][94][95].…”

Comparison of Pulegone and Estragole Chemotypes Provides New Insight Into Volatile Oil Biosynthesis of Agastache rugosa

“…Therefore, studying the formation and mechanism of chemotypes plays an important role in the utilization of A. rugosa . However, only LS and PR in the pulegone biosynthesis pathway has been characterized in A. rugosa ( Maruyama et al, 2002 ; Liu C. et al, 2021 ). In this study, we compared the morphological differences, metabolite constituents, and transcriptomic data between the two chemotypes of A. rugosa .…”

“…The suspension was transferred to an ultrasonic cell disruptor system and ultrasonically disrupted for 25 s each with 35 s on ice between disruptions. The mixture was centrifuged for 30 min at 4°C and 12,000 rpm and the supernatant was carefully removed ( Liu C. et al, 2021 ).…”

Mentha piperita: Essential Oil and Extracts, Their Biological Activities, and Perspectives on the Development of New Medicinal and Cosmetic Products

“…(−)-Menthone, the second most abundant monoterpenoid in the essential oil of M. piperita, is a critical intermediate in menthol biosynthesis and possesses a minty aroma and various acknowledged biological activities. (−)-Menthone is directly synthesized from (+)-pulegone through the reduction of the C2-C8 alkene double bond, catalyzed using (+)-pulegone reductase [76]. (+)-Pulegone is the precursor of (−)-menthone, (−)-menthol, and (+)-menthofuran [58,76].…”