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

• Published in: Biochemical journal Vol. 477; no. 15; p. 2791
• Main Authors: Akere, Aishat, Chen, Serena H, Liu, Xiaohan, Chen, Yanger, Dantu, Sarath Chandra, Pandini, Alessandro, Bhowmik, Debsindhu, Haider, Shozeb
• Format: Journal Article
• Language: English
• Published: England 14.08.2020
• Subjects: Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Deep Learning; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Glycosyltransferases - chemistry; Glycosyltransferases - genetics; Glycosyltransferases - metabolism; Models, Molecular; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Protein Engineering - methods; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Structural Homology, Protein; Structure-Activity Relationship; Substrate Specificity; deep learning; glycosyltransferases; mass spectrometry; molecular dynamics
• ISSN: 1470-8728
• DOI: 10.1042/bcj20200477

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.