Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis
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...
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Frontiers Media S.A.
2021
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oai:doaj.org-article:69ddef3a290c4cd9b4c42a047c4f9acf2021-12-01T19:46:09ZFunctional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis1664-462X10.3389/fpls.2021.780970https://doaj.org/article/69ddef3a290c4cd9b4c42a047c4f9acf2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fpls.2021.780970/fullhttps://doaj.org/toc/1664-462XMonoterpenoids 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.Chanchan LiuQiyu GaoZhuo ShangJian LiuSiwei ZhouJingjie DangLicheng LiuIris LangeNarayanan SrividyaB. Markus LangeQinan WuQinan WuWei LinWei LinWei LinWei LinFrontiers Media S.A.articlepulegone reductasestereoselectivitymolecular dynamics simulationsmenthol biosynthesisMentha piperitaPlant cultureSB1-1110ENFrontiers in Plant Science, Vol 12 (2021) |
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| collection |
DOAJ |
| language |
EN |
| topic |
pulegone reductase stereoselectivity molecular dynamics simulations menthol biosynthesis Mentha piperita Plant culture SB1-1110 |
| spellingShingle |
pulegone reductase stereoselectivity molecular dynamics simulations menthol biosynthesis Mentha piperita Plant culture SB1-1110 Chanchan Liu Qiyu Gao Zhuo Shang Jian Liu Siwei Zhou Jingjie Dang Licheng Liu Iris Lange Narayanan Srividya B. Markus Lange Qinan Wu Qinan Wu Wei Lin Wei Lin Wei Lin Wei Lin Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis |
| description |
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. |
| format |
article |
| author |
Chanchan Liu Qiyu Gao Zhuo Shang Jian Liu Siwei Zhou Jingjie Dang Licheng Liu Iris Lange Narayanan Srividya B. Markus Lange Qinan Wu Qinan Wu Wei Lin Wei Lin Wei Lin Wei Lin |
| author_facet |
Chanchan Liu Qiyu Gao Zhuo Shang Jian Liu Siwei Zhou Jingjie Dang Licheng Liu Iris Lange Narayanan Srividya B. Markus Lange Qinan Wu Qinan Wu Wei Lin Wei Lin Wei Lin Wei Lin |
| author_sort |
Chanchan Liu |
| title |
Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis |
| title_short |
Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis |
| title_full |
Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis |
| title_fullStr |
Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis |
| title_full_unstemmed |
Functional Characterization and Structural Insights Into Stereoselectivity of Pulegone Reductase in Menthol Biosynthesis |
| title_sort |
functional characterization and structural insights into stereoselectivity of pulegone reductase in menthol biosynthesis |
| publisher |
Frontiers Media S.A. |
| publishDate |
2021 |
| url |
https://doaj.org/article/69ddef3a290c4cd9b4c42a047c4f9acf |
| work_keys_str_mv |
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