Abstract
Glycosylation in natural product metabolism and xenobiotic detoxification often leads to disaccharide-modified metabolites. The chemical synthesis of such glycosides typically separates the glycosylation steps in space and time. The option to perform the two-step glycosylation in one pot, and catalyzed by a single permissive enzyme, is interesting for a facile access to disaccharide-modified products. Here, we reveal the glycosyltransferase GT1 from Bacillus cereus (BcGT1; gene identifier: KT821092) for iterative O-β-glucosylation from uridine 5′-diphosphate (UDP)-glucose to form a β-linked disaccharide of different metabolites, including a C15 hydroxylated detoxification intermediate of the agricultural herbicide cinmethylin (15HCM). We identify thermodynamic and kinetic requirements for the selective formation of the disaccharide compared to the monosaccharide-modified 15HCM. As shown by NMR and high-resolution MS, β-cellobiosyl and β-gentiobiosyl groups are attached to the aglycone’s O15 in a 2:1 ratio. Glucosylation reactions on methylumbelliferone and 4-nitrophenol involve reversible glycosyl transfer from and to UDP as well as UDP-glucose hydrolysis, both catalyzed by BcGT1. Collectively, this study delineates the iterative β-d-glucosylation of aglycones by BcGT1 and demonstrates applicability for the programmable one-pot synthesis of disaccharide-modified 15HCM.
Highlights
Glycosylation is a widespread type of chemical conjugation performed on small molecules in natural product biosynthesis[1−3] and metabolite detoxification.[1,3]
The major target of our inquiry was 15-hydroxy cinmethylin (15HCM; 1, Figure 1), which is phase I detoxification metabolite of the pre-emergence herbicide cinmethylin (CM; 2, Figure 1).[22−24] CM is a benzyl ether derivative of the natural terpene 1,4-cineole that is currently used in a commercialized product (Luximax) for integrated weed and grass management.[22,25]
We identified the glycosyltransferase GT1 from Bacillus cereus (BcGT1; GenBank identifier: KT821092) for iterative O-β-glucosylation from uridine 5′-diphosphate (UDP)-glucose to build β-linked disaccharide on different metabolites (15HCM, 1; 4-methylumbelliferone, 4MU, 3; 4nitro-phenol, 4NP, 4; Figure 1)
Summary
Glycosylation is a widespread type of chemical conjugation performed on small molecules in natural product biosynthesis[1−3] and metabolite detoxification.[1,3] Attachment of sugar residue(s) increases the solubility, often defines the bioactivity, and directs the cellular targeting of the metabolite.[4,5] In biology, the task of glycosylation is handled by a class of sugar nucleotide-dependent glycosyltransferases.[4,5] These enzymes use sugar nucleotides as donors for glycosyl transfer to acceptors.[4,5] Glycosyltransferases offer precise α/β stereocontrol of the glycosylation but differ widely in their substrate scope.[4−6] For example, detoxifying glycosyltransferases are often highly permissive regarding the acceptor substrates used.[3,4] Assayed in vitro, some glycosyltransferases of secondary metabolism can use a large diversity of donor and acceptor substrates.[3,7] Due to the interplay of different glycosyltransferases in biosynthesis, the glycosylation on small molecules can give rise to considerable structural diversity.[8,9] Products can be glycosylated at multiple positions, exhibit a disaccharide, or even an oligosaccharide, attached to the aglycone, or feature both modifications at the same time.[8,9] Among the natural products, many (e.g., antibiotics like vancomycin;[10,11] flavonoids like quercetin or luteolin;[12,13] fragrances and flavors like geraniol1,5,14) are found in different glycoside forms and show modulation in function or potency due to change in glycosylation pattern. The steviol glycosides imparting intense sweetness to extracts of the Stevia plant are bis-glycosides, with a disaccharide (stevioside) or trisaccharide (rebaudioside A) attached to the diterpene aglycone.[15,16] Our interest here was on metabolite glycosylation with a disaccharide unit, which is chemically challenging to install and not well explored for glycoside synthesis It prompted a study on glycosyltransferase-catalyzed preparation of disaccharide-modified xenobiotics, with the purpose of establishing a facile and broadly applicable route for metabolite neoglycosylation.[10,17] Besides being widespread in natural products, glycosylation with simple β-D-glucose disaccharides (e.g., β-gentiobiosyl, β-D-glucosyl-(1→6)-β-D-glucosyl) plays a significant role in the detoxification of herbicides (e.g., Picloram;[18] Diphenamid;19 3-phenoxy benzoic acid[20,21] derived from pyrethroids) in plants. The major target of our inquiry was 15-hydroxy cinmethylin (15HCM; 1, Figure 1), which is phase I detoxification metabolite of the pre-emergence herbicide cinmethylin (CM; 2, Figure 1).[22−24] CM is a benzyl ether derivative of the natural terpene 1,4-cineole that is currently used in a commercialized product (Luximax) for integrated weed and grass management.[22,25] Besides their importance as analytical reference, disaccharide glucosides of 15HCM have interest for the evaluation of biological efficacy
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