期刊
PLANT JOURNAL
卷 111, 期 6, 页码 1539-1549出版社
WILEY
DOI: 10.1111/tpj.15904
关键词
crystal structure; dhurrin; glycosyltransferase; prunasin; rational engineering; sambunigrin; taxiphyllin
资金
- Sapere Aude Starting Grant from the Independent Research Fund Denmark [7026-00041B]
- Novo Nordisk Foundation Emerging Investigator Grant [NNF19OC0055356]
- Novo Nordisk Foundation [NNF18OC0034744, NNF10CC1016517, NNF20CC0035580]
- VILLUM Foundation Young Investigator Program fellowship [13167]
- Danish Agency for Science, Technology, and Innovation [7129-00003B]
This study reports the substrate-bound crystal structures and rational engineering of UGT85B1 from Sorghum bicolor involved in the biosynthesis of cyanogenic glucoside dhurrin. By combining mutations and substitution of specific residues, the researchers successfully shifted the substrate and stereo-specificities of the enzyme. The findings improve our understanding of UGTs involved in the biosynthesis of cyanogenic glucosides and have potential applications in biotechnology.
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 angstrom 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.
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