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

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.

Quantification and Localization of Formylated Phloroglucinol Compounds (FPCs) in Eucalyptus Species.

The Eucalyptus genus is a hyper-diverse group of long-lived trees from the Myrtaceae family, consisting of more than 700 species. Eucalyptus are widely distributed across their native Australian landscape and are the most widely planted hardwood forest trees in the world. The ecological and economic success of Eucalyptus trees is due, in part, to their ability to produce a plethora of specialized metabolites, which moderate abiotic and biotic interactions. Formylated phloroglucinol compounds (FPCs) are an important class of specialized metabolites in the Myrtaceae family, particularly abundant in Eucalyptus. FPCs are mono- to tetra-formylated phloroglucinol based derivatives, often with an attached terpene moiety. These compounds provide chemical defense against herbivory and display various bioactivities of pharmaceutical relevance. Despite their ecological and economic importance, and continued improvements into analytical techniques, FPCs have proved challenging to study. Here we present a simple and reliable method for FPCs extraction, identification and quantification by UHPLC-DAD-ESI-Q-TOF-MS/MS. The method was applied to leaf, flower bud, and flower samples of nine different eucalypt species, using a small amount of plant material. Authentic analytical standards were used to provide high resolution mass spectra and fragmentation patterns. A robust method provides opportunities for future investigations into the identification and quantification of FPCs in complex biological samples with high confidence. Furthermore, we present for the first time the tissue-based localization of FPCs in stem, leaf, and flower bud of Eucalyptus species measured by mass spectrometry imaging, providing important information for biosynthetic pathway discovery studies and for understanding the role of those compounds in planta.