TY - JOUR
T1 - Rational design of resveratrol o-methyltransferase for the production of pinostilbene
AU - Herrera, Daniela P.
AU - Chánique, Andrea M.
AU - Martínez-Márquez, Ascensión
AU - Bru-Martínez, Roque
AU - Kourist, Robert
AU - Parra, Loreto P.
AU - Schüller, Andreas
N1 - Funding Information:
Funding: This research was funded by PROYECTO INTERDISCIPLINA-VRI-UC-II160020, number 3514-913, Pontificia Universidad Católica de Chile (to L.P.P. and A.S.) and BECA DE DOCTORADO NACIONAL 2016, number 21161084, National Agency for Research and Development (ANID), Chile (to D.P.H.), for which we are grateful. The APC was funded by Pontificia Universidad Católica de Chile.
Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
PY - 2021/5/1
Y1 - 2021/5/1
N2 - 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.
AB - 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.
KW - Enzyme engineering
KW - O-methyltransferases
KW - Pinostilbene
KW - Protein models
KW - Stilbenes
KW - Substrate selectivity
UR - http://www.scopus.com/inward/record.url?scp=85104488097&partnerID=8YFLogxK
U2 - 10.3390/ijms22094345
DO - 10.3390/ijms22094345
M3 - Article
C2 - 33919396
AN - SCOPUS:85104488097
SN - 1661-6596
VL - 22
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 9
M1 - 4345
ER -