TY - JOUR

T1 - Structure-guided engineering of key amino acids in UGT85B1 controlling substrate and stereo-specificity in aromatic cyanogenic glucoside biosynthesis

AU - Del Giudice, Rita

AU - Putkaradze, Natalia

AU - dos Santos, Bruna Marques

AU - Hansen, Cecilie Cetti

AU - Crocoll, Christoph

AU - Motawia, Mohammed Saddik

AU - Fredslund, Folmer

AU - Laursen, Tomas

AU - Welner, Ditte Hededam

N1 - Funding Information: This work was supported by a Sapere Aude Starting Grant from the Independent Research Fund Denmark (7026‐00041B) and a Novo Nordisk Foundation Emerging Investigator Grant (NNF19OC0055356) to TL and by Novo Nordisk Foundation grants (NNF18OC0034744, NNF10CC1016517, and NNF20CC0035580). CCH was supported by a PhD fellowship provided through a VILLUM Foundation Young Investigator Program fellowship granted to Elizabeth H. J. Neilson (grant number 13167). We thank the Danish Agency for Science, Technology, and Innovation for funding the instrument center DanScatt (7129‐00003B ). Publisher Copyright: © 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

PY - 2022

Y1 - 2022

N2 - Cyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP-dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure–function relationships controlling scope and specificity remain poorly understood. Here, we report substrate-bound crystal structures and rational engineering of substrate and stereo-specificities of UGT85B1 from Sorghum bicolor involved in biosynthesis of the cyanogenic glucoside dhurrin. Substrate specificity was shifted from the natural substrate (S)-p-hydroxymandelonitrile to (S)-mandelonitrile by combining a mutation to abolish hydrogen bonding to the p-hydroxyl group with a mutation to provide steric hindrance at the p-hydroxyl group binding site (V132A/Q225W). Further, stereo-specificity was shifted from (S) to (R) by substituting four rationally chosen residues within 6 Å of the nitrile group (M312T/A313T/H408F/G409A). These activities were compared to two other UGTs involved in the biosynthesis of aromatic cyanogenic glucosides in Prunus dulcis (almond) and Eucalyptus cladocalyx. Together, these studies enabled us to pinpoint factors that drive substrate and stereo-specificities in the cyanogenic glucoside biosynthetic UGTs. The structure-guided engineering of the functional properties of UGT85B1 enhances our understanding of the evolution of UGTs involved in the biosynthesis of cyanogenic glucosides and will enable future engineering efforts towards new biotechnological applications.

AB - Cyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP-dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure–function relationships controlling scope and specificity remain poorly understood. Here, we report substrate-bound crystal structures and rational engineering of substrate and stereo-specificities of UGT85B1 from Sorghum bicolor involved in biosynthesis of the cyanogenic glucoside dhurrin. Substrate specificity was shifted from the natural substrate (S)-p-hydroxymandelonitrile to (S)-mandelonitrile by combining a mutation to abolish hydrogen bonding to the p-hydroxyl group with a mutation to provide steric hindrance at the p-hydroxyl group binding site (V132A/Q225W). Further, stereo-specificity was shifted from (S) to (R) by substituting four rationally chosen residues within 6 Å of the nitrile group (M312T/A313T/H408F/G409A). These activities were compared to two other UGTs involved in the biosynthesis of aromatic cyanogenic glucosides in Prunus dulcis (almond) and Eucalyptus cladocalyx. Together, these studies enabled us to pinpoint factors that drive substrate and stereo-specificities in the cyanogenic glucoside biosynthetic UGTs. The structure-guided engineering of the functional properties of UGT85B1 enhances our understanding of the evolution of UGTs involved in the biosynthesis of cyanogenic glucosides and will enable future engineering efforts towards new biotechnological applications.

KW - crystal structure

KW - dhurrin

KW - glycosyltransferase

KW - prunasin

KW - rational engineering

KW - sambunigrin

KW - taxiphyllin

U2 - 10.1111/tpj.15904

DO - 10.1111/tpj.15904

M3 - Journal article

C2 - 35819080

AN - SCOPUS:85135266375

VL - 111

SP - 1539

EP - 1549

JO - Plant Journal

JF - Plant Journal

SN - 0960-7412

IS - 6

ER -