Exploring the Nucleophilic N- and S-Glycosylation Capacity ofBacillus licheniformisYjiC Enzyme

Glycosylation is a well-characterized glycosyltransferase (GT) enzyme-catalyzed reaction, in cells, involved in metabolism, cell integrity, molecular recognition and pathogenicity, and post-modification of secondary metabolites during biosynthesis [1][2][3][4].GTs are ubiquitous in nature and transfer sugar moieties from activated nucleotide diphosphate sugars (NDP-D/L-sugars) to acceptor molecules.Leloir GTs are NDP-sugar dependent and transfer sugar units to lipid, nucleic acid, natural products, and other small molecules at nucleophilic oxygen (O-), nitrogen (N-), sulfur (S-), or carbon (C-).According to the recent CAZy classification (http://www.cazy.org/),GTs are classified into 110 different families.Among them, GT1 family proteins are inverting enzymes having GT-B type 3D structure transferring diverse sugars to small molecules [5,6].The glycosylation of natural products (NPs) influences the physical, chemical and biological properties of the parent molecules.Especially, the sugar conjugation to therapeutically important NPs alters the pharmacological and pharmacokinetic properties including water solubility, stability, specificity, as well as biological actions of the compounds [7,8].Due to the emerging resistance to the different therapeutics, recent research has been focused on designing/ developing bioactive molecules by modification of previously known compounds using various approaches such as by applying microbial enzymes and cells as biocatalysts.Glycosylation is one of the most prominent tools to create glycoside libraries of bioactive small molecules as glycosylation results in an alteration in the pharmacokinetic properties of the parent compounds [9].In this context, the search for novel glycosyltransferases with tolerance to diverse sets of donor substrates and acceptor compounds is expanding in importance.GTs from various organisms have been used to glucosylate diverse sets of plant natural products, specifically flavonoids [10,11].In this study, we have investigated the application of a GT, YjiC from non-pathogenic Bacillus licheniformis DSM 13 strain for glycosylation of various industrially important amino (NH 2 ) and thiol (SH) functional groupcontaining acceptor substrates.YjiC has been extensively studied for its donor and acceptor substrate promiscuity towards nucleophilic O-glycosylation of diverse sets of natural products using NDP-D/L-sugars as donor substrates [12,13].Nucleophilic N-, S-, C-glycosylation is regarded as rare in comparison to O-glycosylation of natural products.This puts the emphasis on research with those GTs capable of not only O-glycosylation but also able to generate other natural products with uncommon glycosidic linkages.GTs able to form C-C glycosydic linkages are gaining attention because of the stability of C-C bonds, and the resulting activity of both glycosyl and aglycone parts [14].Likewise, N-and S-linked glycosidic linkages are also equally important for developing novel natural products with potential biological activity.In this study, we explored promiscuous YjiC enzyme for N-and YjiC, a glycosyltransferase from Bacillus licheniformis, is a well-known versatile enzyme for glycosylation of diverse substrates.Although a number of O-glycosylated products have been produced using YjiC, no report has been updated for nucleophilic N-, S-, and Cglycosylation.Here, we report the additional functional capacity of YjiC for nucleophilic Nand Sglycosylation using a broad substrate spectrum including UDP-α-D-glucose, UDP-N-acetyl glucosamine, UDP-N-acetylgalactosamine, UDP-α-D-glucuronic acid, TDP-α-L-rhamnose, TDP-α-D-viosamine, and GDP-α-Lfucose as donor and various amine and thiol groups containing natural products as acceptor substrates.The results revealed YjiC as a promiscuous enzyme for conjugating diverse sugars at amine and thiol functional groups of small molecules applicable for generating glycofunctionalized chemical diversity libraries.The glycosylated products were analyzed using HPLC and LC/MS and compared with previous reports.