Exploration Of OsSGT2-catalyzed Testosterone Glycodiversification And The Residual Substitutions To Improve N-acetylglucosaminylation Of Testosterone

Secondary metabolites containing glycosyl groups are an important source of new drug discovery.As key elements of these natural pharmacophores,attached sugars are often critical for biological activity.Glycosylation is a key step in the synthesis of structurally diverse glycosides,which can improve the medicinal properties and bioavailability of natural drugs.Glycodiversification is developing as one of the most important catalytic tools for creating biologically active small molecule glycosides libraries,but many problems remain to be solved.For example,the chemical synthesis of glycosides requires glycosyl activation and involves multiple protection/de-protection steps to control regional selectivity,which tends to increase cost.Therefore,mining a green and efficient glycosyl diversification tool has become a research hotspot in this field.Glycosyltransferases(UGTs)are a class of superfamly enzymes that catalyze glycosylation by transferring glycosyl groups from UDP-sugars(glycosyl donors)to acceptors(glycosyl receptors),demonstrating high efficiency and stereoselectivity/regioselectivity for the synthesis of glycosides in a green and efficient way.Previous chemical composition studies showed that there were a large number of steroidal glycosides in Ornithogalum saundersiae,and it was speculated that the plant had glycosyltransferases involved in the biosynthesis of glycoside products.In this study,OsSGT2,a glycosyltransferase with testosterone glycosyldiversification ability,was identified from Ornithogalum saundersiae by transcriptome analysis and gene mining.In this paper,the catalytic function and enzymatic properties of OsSGT2 as well as the catalytic efficiency of UDP-N-acetylglucosamine improved by mutation modification were studied.1-Study on glycodiversification of testosteroneThe diversity expansion of testosterone 17-O-β-glycosides(TGs)would increase the probability to screen more active molecules from their acetylated derivatives with anticancer activities.Glycosyltransferases(GTs)responsible for the increased diversity of TGs,however,were seldom documented.Herein,a glycosyltransferase OsSGT2 with a capacity of testosterone glycodiversification was identified from Ornithogalum saundersiae through transcriptome-wide mining and comprehensive exploration.Specifically,OsSGT2 was demonstrated to be reactive with testosterone and 8 donors.Furthermore,OsSGT2 displayed both sugar-aglycon and sugar-sugar GT activities.Under the action of OsSGT2,testosterone glycodiversification could be achieved,generating testosterone monoglycosides and biosides with varied conversion rates.The structures of 6 compounds were identified by high performance liquid chromatography,ultraviolet absorption spectra,high resolution mass spectrometry and nuclear magnetic resonance spectroscopy,and 3 of them were new compounds.In addition,we also found that OsSGT2 displayed sugar-aglycon,sugar-sugar glycosyltransferase and specific galactosidase activities,and optimized the glycosylation reaction conditions and determined the enzymatic kinetics parameters.2.Site-directed mutation increasing the catalytic efficiency of UDP-GlcNAcAmong the 8 donors,the conversion of UDP-Glc was the highest,approaching 90%,while the conversion rates of UDP-G1cNAc,UDP-Gal,helicin and UDP-Rha were less than 10%.A residue F395 was predicted by molecule docking to contribute to the conversion enhancement of UDP-GlcNAc.Saturated mutagenesis on F395 was thus performed with the aim to improve the conversion of UDP-GlcNAc.Eight variants displayed increased conversions and the mutant F395C got the highest conversion of 72.11±7.82%,8 times than that of the wild-type.The conversion enhancement was attributed to the reduced steric hindrance and the more stable conformation of UDPGlcNAc.This study provides a promising alternative for diversity expansion of TGs,also significant insights into the molecular basis for the conversion improvement of sugar donors.3.OsSGT2 catalyzing the glycodiversification of other acceptorsBesides OsSGT2 catalytic reaction of testosterone and at least eight kinds of donors,we used 31 accptors and 33 glycosyl donors to study the promiscuity.In combination with high performance liquid chromatography(HPLC),ultraviolet absorption spectrum,high resolution mass spectrometry and nuclear magnetic resonance spectrum,we identified the structure of the glycosylated products.The results show that OsSGT2 can catalyze the glycodiversification of bufalin,fisetin,abiraterone and(R/S)-protopanaxatriol to generate the corresponding glycoside products,which also confirms that OsSGT2 has both acceptor and donor promiscuities,providing a powerful component for biosynthesis of glycosides.