Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases.

Aishat Akere,Xiaohan Liu,Alessandro Pandini,Sarath Chandra Dantu,Serena H Chen,Debsindhu Bhowmik,Yanger Chen,Shozeb Haider

doi:10.1042/bcj20200477

Abstract

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.

Highlights

Glycosyltransferases (GTs) are a large family of structurally conserved enzymes responsible for catalyzing the transfer of a sugar moiety from an activated donor sugar to an acceptor molecule [1]
There are a total of 122 Arabidopsis thaliana UDP-dependent glycosyltransferases (UGT) genes, which are grouped into 14 phylogenetic groups A-N (Supplementary Figure S1) [6]
We report on the qualitative substrate specificities of 76E1, 76E2, 76E4, 76E5, and 76D1 Group H A. thaliana UGTs using mass spectrometry (MS) based methods

Summary

Introduction

Glycosyltransferases (GTs) are a large family of structurally conserved enzymes responsible for catalyzing the transfer of a sugar moiety from an activated donor sugar to an acceptor molecule [1]. Glycosylation of plant secondary metabolites is catalyzed by uridine diphosphate (UDP) glycosyltransferases (UGTs) belonging to family 1 GTs [3]. These plant secondary metabolites include alkaloids, terpenoids, flavonoids, isoflavonoids, and other small molecules. The planet-produced enzymes like ELELYSO (Protalix BioTherapeutics), is central to the production of lysosomal human GBA enzyme in carrot cells. Another human lysosomal hydrolase is α-galactosidase A, used for treating Fabry disease [5]. There are a total of 122 Arabidopsis thaliana UGT genes, which are grouped into 14 phylogenetic groups A-N (Supplementary Figure S1) [6]

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Discussion

Conclusion