Glucosylation of (±)-Menthol by Uridine-Diphosphate-Sugar Dependent Glucosyltransferases from Plants

Abstract

Menthol is a cyclic monoterpene alcohol of the essential oils of plants of the genus Mentha, which is in demand by various industries due to its diverse sensorial and physiological properties. However, its poor water solubility and its toxic effect limit possible applications. Glycosylation offers a solution as the binding of a sugar residue to small molecules increases their water solubility and stability, renders aroma components odorless and modifies bioactivity. In order to identify plant enzymes that catalyze this reaction, a glycosyltransferase library containing 57 uridine diphosphate sugar-dependent enzymes (UGTs) was screened with (±)-menthol. The identity of the products was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Five enzymes were able to form (±)-menthyl-β-d-glucopyranoside in whole-cell biotransformations: UGT93Y1, UGT93Y2, UGT85K11, UGT72B27 and UGT73B24. In vitro enzyme activity assays revealed highest catalytic activity for UGT93Y1 (7.6 nkat/mg) from Camellia sinensis towards menthol and its isomeric forms. Although UGT93Y2 shares 70% sequence identity with UGT93Y1, it was less efficient. Of the five enzymes, UGT93Y1 stood out because of its high in vivo and in vitro biotransformation rate. The identification of novel menthol glycosyltransferases from the tea plant opens new perspectives for the biotechnological production of menthyl glucoside.

Keywords: Camellia sinensis; UDP-glucosyltransferase; UGT93Y1; menthol; menthyl glucoside.

Figure 1

Relative biotransformation rate of UGT72B27, UGT73B24, UGT85K11, UGT93Y1 and UGT93Y2 in % towards (±)-menthol (A) and (−)-menthol (B). The intensity of the pseudo-molecular ion m/z 363 [M + HCOO]⁻ of menthyl glucoside was determined, and the UGT with the highest value was set to 100%. Samples were analyzed after 6, 24 and 48 h after substrate addition. pGEX-4T-1 served as negative control.

Figure 2

Qualitative in vitro substrate screening of UGT93Y1 with (±)-menthol (A), (1S,2R,5R)-(+)-isomenthol (B), (1S,2S,5R)-(+)-neomenthol (C) and fenchyl alcohol (D) by LC-MS. Selected ion chromatograms (m/z 363 and 361), mass spectra in negative mode (-MS) and product ion spectra of the pseudo-molecular ions (m/z 363 and 361; -MS2) are shown.

Figure 3

Determination of the relative enzymatic activities with UDP Glo™ Glycosyltransferase assay (A) and UGT93Y1 enzyme kinetics for (±)-menthol (B). The relative enzyme activity was calculated after measuring the release of UDP during the glucosylation reaction. The highest enzymatic activity was set at 100%, and the relative enzyme activities calculated (A). The enzyme activity versus substrate concentration graph for UGT93Y1 from C. sinensis var. sinensis was determined using the UDP Glo™ Glycosyltransferase assay. The calculation was performed with KaleidaGraph (https://www.synergy.com/; v4.5.4; accessed on 8 September 2021).

Figure 4

¹³C NMR analysis of biotransformation products formed from (±)-menthol by UGT93Y1 whole-cell biocatalysis. NMR spectrum of products produced from (±)-menthol (A). Chemical shifts (δ) in ppm of (−)- and (+)-menthyl-β-d-glucopyranoside taken from [42] of (+)-menthol from [1] and of the reaction products (B). Chemical structures of (−)- and (+)-menthyl-β-d-glucopyranoside (C).

Figure 5

Homology modelling. Prediction of the 3D structure of UGT93Y1 was performed by the IntFOLD Integrated Protein Structure and Function Prediction Server (https://www.reading.ac.uk/bioinf/IntFOLD/; accessed on 8 September 2021) with default values. The result was visualized with UCSF Chimera (https://www.cgl.ucsf.edu/chimera/; accessed on 8 September 2021), and ligand docking was performed with the Autodock/Vina tool (http://vina.scripps.edu/download.html; accessed on 8 September 2021). Predicted 3D structure of UGT93Y1 (A), predicted (−)-menthol and (+)-menthol pose in the active site of UGT93Y1 (trichlorophenol pose from 2vce (https://www.rcsb.org/; accessed on 8 September 2021) is shown for comparison) (B) and ligand–protein interactions predicted by Discovery Studio Visualizer v19.1.0.18287 (https://discover.3ds.com/discovery-studio-visualizer-download; accessed on 8 September 2021) (C).