Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene.

Roque Bru-Martínez,Andrea M Chánique,Andreas Schüller,Loreto P Parra,Robert Kourist,Ascensión Martínez-Márquez,Daniela P Herrera

doi:10.3390/ijms22094345

Abstract

Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol’s enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.

Highlights

Stilbenes are phenolic compounds derived from the secondary metabolism of plants that participate in their constitutive and inducible defense mechanisms [1]
We focused on enzymes that accept stilbenes to get an insight into their distribution among different OMT-I
We focused on two regions to perform the rational design to modify the substrate selectivity of VvROMT

Summary

Introduction

Stilbenes are phenolic compounds derived from the secondary metabolism of plants that participate in their constitutive and inducible defense mechanisms [1]. Resveratrol (trans3,5,40 -trihydroxystilbene) is one of the most studied stilbenes due to its health-promoting properties with anticancer [2], antioxidant [3], anti-inflammatory [4,5], cardioprotective [6], and neuroprotective activities [7]. Despite these properties, resveratrol has less than 1% of oral bioavailability in humans [8], an 8–14 min half-life [9,10], and undergoes rapid metabolization due to glucuronidation, sulfation, and hydrogenation reactions of its aliphatic double bond [11]. It has been shown that protection of resveratrol’s 3 and 5-hydroxyl moieties by O-methylation increases lipophilicity, stability, uptake into human cells, and its bioavailability [13,14]

Methods

Results

Conclusion