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

• Published in: The Plant journal : for cell and molecular biology 2022-09, Vol.111 (6), p.1539-1549
• Main Authors: Del Giudice, Rita, Putkaradze, Natalia, Santos, Bruna Marques, Hansen, Cecilie Cetti, Crocoll, Christoph, Motawia, Mohammed Saddik, Fredslund, Folmer, Laursen, Tomas, Welner, Ditte Hededam
• Format: Article
• Language: English
• Subjects: Amino acids; Binding sites; Biosynthesis; Biotechnology; Crystal structure; dhurrin; Engineering; Eucalyptus; Eucalyptus cladocalyx; Glucosides; Glycosylation; Glycosyltransferase; Hydrogen bonding; Hydroxyl groups; Mandelonitrile; Mutation; prunasin; Prunus dulcis; rational engineering; sambunigrin; Sorghum; Steric hindrance; Structural stability; Structure-function relationships; Substrate specificity; Substrates; taxiphyllin
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.15904

SUMMARY 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. Significance Statement Glycosylation of plant natural products is an important modification that enhances their stability and solubility and prevents autotoxicity. Understanding the structure–function relationship of glycosyltransferases is critical for our understanding of plant natural product biosynthesis. We provide structural insights into the features controlling substrate and stereo‐specificity of UGT85B1, the glycosyltransferase catalyzing the production of the cyanogenic glucoside dhurrin in Sorghum bicolor. This study will enable engineering of UGT85 enzymes for tailored glycosylation of a palette of natural products.