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Aloesone is a bioactive natural product and biosynthetic precursor of rare glucosides found in rhubarb and some aloe plants including Aloe vera. This study aimed to investigate biocatalytic aloesone glycosylation and more than 400 uridine diphosphate-dependent glycosyltransferase (UGT) candidates, including multifunctional and promiscuous enzymes from a variety of plant species were assayed. As a result, 137 selective aloesone UGTs were discovered, including four from the natural producer rhubarb. Rhubarb UGT72B49 was further studied and its catalytic constants (k cat = 0.00092 ± 0.00003 s−1, K M = 30 ± 2.5 μM) as well as temperature and pH optima (50 °C and pH 7, respectively) were determined. We further aimed to find an efficient aloesone glycosylating enzyme with potential application for biocatalytic production of the glucoside. We discovered UGT71C1 from Arabidopsis thaliana as an efficient aloesone UGT showing a 167-fold higher catalytic efficiency compared to that of UGT72B49. Interestingly, sequence analysis of all the 137 newly identified aloesone UGTs showed that they belong to different phylogenetic groups, with the highest representation in groups B, D, E, F and L. Finally, our study indicates that aloesone C -glycosylation is highly specific and rare, since it was not possible to achieve in an efficient manner with any of the 422 UGTs assayed, including multifunctional GTs and 28 known C -UGTs. [ABSTRACT FROM AUTHOR]

C 13 -apocarotenoids (norisoprenoids) are carotenoid-derived oxidation products that perform important physiological functions in plants. Although their biosynthetic pathways have been extensively studied, their metabolism including glycosylation remains poorly understood. Candidate uridine-diphosphate glycosyltransferase genes ( UGTs ) were selected based on their high transcript abundance in comparison with other UGTs in vegetative tissues of Nicotiana benthamiana and peppermint ( Mentha × piperita ), as these tissues are rich sources of apocarotenoid glucosides. Hydroxylated C 13 -apocarotenol substrates were produced by P450-catalyzed biotransformation and microbial/plant enzyme systems were established for the synthesis of glycosides. Natural substrates were identified by physiological aglycone libraries prepared from isolated plant glycosides. In total, we identified six UGTs that catalyze the glucosylation of C 13 -apocarotenols, where Glc is bound either to the cyclohexene ring or the butane side chain. MpUGT86C10 is a superior novel enzyme that catalyzes the glucosylation of allelopathic 3-hydroxy- α -damascone, 3-oxo- α -ionol, 3-oxo-7,8-dihydro- α -ionol (Blumenol C), and 3-hydroxy-7,8-dihydro- β -ionol, whereas a germination test demonstrated the higher phytotoxic potential of a norisoprenoid glucoside in comparison to its aglycone. Glycosylation of C 13 -apocarotenoids has several functions in plants, including increased allelopathic activity of the aglycone, facilitating exudation by roots and allowing symbiosis with arbuscular mycorrhizal fungi. The results enable in-depth analysis of the roles of glycosylated norisoprenoid allelochemicals, the physiological functions of apocarotenoids during arbuscular mycorrhizal colonization, and the associated maintenance of carotenoid homeostasis., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Bacterial P450s have considerable potential for biotechnological applications. The P450 CYP106A2 from Bacillus megaterium ATCC 13368 converts progesterone to several hydroxylated products that are important precursors for pharmaceutical substances. As high yields of monohydroxylated products are required for biotechnological processes, improving this conversion is of considerable interest. It has previously been shown that the binding mode of the redox partner can affect the selectivity of the progesterone hydroxylation, being more stringent in case of the Etp1 compared with Adx(4-108). Therefore, in this study we aimed to improve hydroxylation selectivity by optimizing the binding of Adx(4-108) with CYP106A2 allowing for a shorter distance between both redox centers. To change the putative binding interface of Adx(4-108) with CYP106A2, molecular docking was used to choose mutation sites for alteration. Mutants at positions Y82 and P108 of Adx were produced and investigated, and confirmed our hypothesis. Protein-protein docking, as well as conversion studies, using the mutants demonstrated that the iron-sulfur(FeS) cluster/heme distance diminished significantly, which subsequently led to an approximately 2.5-fold increase in 15β-hydroxyprogesterone, the main product of progesterone conversion by CYP106A2., (© 2018 Federation of European Biochemical Societies.)