Achievements and impacts of glycosylation reactions involved in natural product biosynthesis in prokaryotes

Bioactive natural products, such as polyketides, flavonoids, glycopeptides, and aminoglycosides, have been used as therapeutic agents. Many of them contain structurally diverse sugar moieties attached to the aglycone core structures. Glycosyltransferases (GTs) catalyze the attachment of nucleotide-activated sugar substrates to acceptor aglycones. Because these sugar moieties are usually essential for biological activity, in vivo pathway engineering in prokaryotic hosts and in vitro enzymatic approaches coupled with GT engineering are currently being used to synthesize novel glycosylated derivatives, and some of them exhibited improved biological activities compared to the parent molecules. Therefore, harnessing the potential of diverse glycosylation reactions in prokaryotes will increase the structural diversity of natural products and the possibility to generate new bioactive products.     

Development of a Streptomyces venezuelae-based combinatorial biosynthetic system for the production of glycosylated derivatives of doxorubicin and its biosynthetic intermediates

Doxorubicin, one of the most widely used anticancer drugs, is composed of a tetracyclic polyketide aglycone and l-daunosamine as a deoxysugar moiety, which acts as an important determinant of its biological activity. This is exemplified by the fewer side effects of semisynthetic epirubicin (4'-epi-doxorubicin). An efficient combinatorial biosynthetic system that can convert the exogenous aglycone ε-rhodomycinone into diverse glycosylated derivatives of doxorubicin or its biosynthetic intermediates, rhodomycin D and daunorubicin, was developed through the use of Streptomyces venezuelae mutants carrying plasmids that direct the biosynthesis of different nucleotide deoxysugars and their transfer onto aglycone, as well as the postglycosylation modifications. This system improved epirubicin production from ε-rhodomycinone by selecting a substrate flexible glycosyltransferase, AknS, which was able to transfer the unnatural sugar donors and a TDP-4-ketohexose reductase, AvrE, which efficiently supported the biosynthesis of TDP-4-epi-l-daunosamine. Furthermore, a range of doxorubicin analogs containing diverse deoxysugar moieties, seven of which are novel rhodomycin D derivatives, were generated. This provides new insights into the functions of deoxysugar biosynthetic enzymes and demonstrates the potential of the S. venezuelae-based combinatorial biosynthetic system as a simple biological tool for modifying structurally complex sugar moieties attached to anthracyclines as an alternative to chemical syntheses for improving anticancer agents.     

Features and applications of bacterial glycosyltransferases: current state and prospects

The bioactivity of many natural products including valuable antibiotics and anticancer therapeutics depends on their sugar moieties. Changes in the structures of these sugars can deeply influence the biological activity, specificity and pharmacological properties of the parent compounds. The chemical synthesis of such sugar ligands is exceedingly difficult to carry out and therefore impractical to establish on a large scale. Therefore, glycosyltransferases are essential tools for chemoenzymatic and in vivo approaches for the development of complex glycosylated natural products. In the last 10 years, several examples of successful alteration and diversification of natural product glycosylation patterns via metabolic pathway engineering and enzymatic glycodiversification have been described. Due to the relaxed substrate specificity of many sugar biosynthetic enzymes and glycosyltransferases involved in natural product biosynthesis, it is possible to obtain novel glycosylated compounds using different methods. In this review, we would like to provide an overview of recent advances in diversification of the glycosylated natural products and glycosyltransferase engineering.     

天然产物的糖基化及糖基侧链改造

抗生素和抗癌药物等多种天然产物的活性都依赖于其糖基侧链,糖基侧链结构的变化对母体化合物的生物活性、底物适应性及药理学性质具有重要影响。糖基侧链结构变化多端,修饰、改变天然产物的糖基侧链已成为获得临床候选药物的重要方法。利用化学法和酶法,研究者创造了多种改造天然产物糖基化的方法。详细介绍了天然产物的糖基化过程,并从组合生物学、糖基转移酶改造、糖类随机化及新型糖类随机化和糖基转移酶可逆性四方面阐述了糖基侧链的改造方法。     

Recent progress in synthesis of carbohydrates with sugar nucleotide-dependent glycosyltransferases

Sugar nucleotide-dependent glycosyltransferases (GTs) are key enzymes that catalyze the formation of glycosidic bonds in nature. They have been increasingly applied in the synthesis of complex carbohydrates and glycoconjugates with or without in situ generation of sugar nucleotides. Human GTs are becoming more accessible and new bacterial GTs have been identified and characterized. An increasing number of crystal structures elucidated for GTs from mammalian and bacterial sources facilitate structure-based design of mutants as improved catalysts for synthesis. Automated platforms have also been developed for chemoenzymatic synthesis of carbohydrates. Recent progress in applying sugar nucleotide-dependent GTs in enzymatic and chemoenzymatic synthesis of mammalian glycans and glycoconjugates, bacterial surface glycans, and glycosylated natural products from bacteria and plants are reviewed.     

Terpene glucoside production: Improved biocatalytic processes using glycosyltransferases

Terpenoids are one of the main classes of natural products. In plants, a large fraction of the terpenoids is present as nonvolatile glycosides. The terpene glycosides have attracted much attention as antimicrobials, flavor precursors, and detergents. They are either extracted from plant materials or are synthesized by chemical and biocatalytic methods. Up to now, biotechnological production of terpene glycosides is based on reversed hydrolysis performed by glycosidases. However, this method suffers from low yields as a matter of principle. Recently, the first uridine diphosphate‐glucose:monoterpenol β‐d ‐glucosyltransferase (GT) genes were cloned and characterized from grapevine (Vitis vinifera) and kiwi (Actinidia deliciosa). Heterologous expression in Escherichia coli yielded promiscuous GT enzymes that efficiently glucosylated primary monoterpenols, simple alcohols, and phenols. The GT enzymes differed in substrate preference and activity toward their terpenoid substrates. Biotransformation experiments confirmed the applicability of the novel GTs in biocatalytic processes for the production of these novel compounds. In the near future, terpene glucosides will become commercially available for food, cosmetic, and pharmaceutical industry due to improved biocatalytic processes involving GT enzymes.     

Sweet antibiotics - the role of glycosidic residues in antibiotic and antitumor activity and their randomization

A large number of antibiotics are glycosides. In numerous cases the glycosidic residues are crucial to their activity; sometimes, glycosylation only improves their pharmacokinetic parameters. Recent developments in molecular glycobiology have improved our understanding of aglycone vs. glycoside activities and made it possible to develop new, more active or more effective glycodrugs based on these findings – a very illustrative recent example is vancomycin. The majority of attention has been devoted to glycosidic antibiotics including their past, present, and probably future position in antimicrobial therapy. The role of the glycosidic residue in the biological activity of glycosidic antibiotics, and the attendant targeting and antibiotic selectivity mediated by glycone and aglycone in antibiotics some antitumor agents is discussed here in detail. Chemical and enzymatic modifications of aglycones in antibiotics, including their synthesis, are demonstrated on various examples, with particular emphasis on the role of specific and mutant glycosyltransferases and glycorandomization in the preparation of these compounds. The last section of this review describes and explains the interactions of the glycone moiety of the antibiotics with DNA and especially the design and structure–activity relationship of glycosidic antibiotics, including their classification based on their aglycone and glycosidic moiety. The new enzymatic methodology 'glycorandomization' enabled the preparation of glycoside libraries and opened up new ways to prepare optimized or entirely novel glycoside antibiotics.     

Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution

Natural products, many of which are decorated with essential sugar residues, continue to serve as a key platform for drug development. Adding or changing sugars attached to such natural products can improve the parent compound's pharmacological properties, specificity at multiple levels, and/or even the molecular mechanism of action. Though some natural-product glycosyltransferases (GTs) are sufficiently promiscuous for use in altering these glycosylation patterns, the stringent specificity of others remains a limiting factor in natural-product diversification and highlights a need for general GT engineering and evolution platforms. Herein we report the use of a simple high-throughput screen based on a fluorescent surrogate acceptor substrate to expand the promiscuity of a natural-product GT via directed evolution. Cumulatively, this study presents variant GTs for the glycorandomization of a range of therapeutically important acceptors, including aminocoumarins, flavonoids and macrolides, and a potential template for engineering other natural-product GTs.     

Expanding substrate specificity of GT-B fold glycosyltransferase via domain swapping and high-throughput screening

Glycosyltransferases (GTs) are crucial enzymes in the biosynthesis and diversification of therapeutically important natural products, and the majority of them belong to the GT-B superfamily, which is composed of separate N- and C-domains that are responsible for the recognition of the sugar acceptor and donor, respectively. In an effort to expand the substrate specificity of GT, a chimeric library with different crossover points was constructed between the N-terminal fragments of kanamycin GT (kanF) and the C-terminal fragments of vancomycin GT (gtfE) genes by incremental truncation method. A plate-based pH color assay was newly developed for the selection of functional domain-swapped GTs, and a mutant (HMT31) with a crossover point (N-kanF-669 bp and 753 bp-gtfE-C) for domain swapping was screened. The most active mutant HMT31 (50 kDa) efficiently catalyzed 2-DOS (aglycone substrate for KanF) glucosylation using dTDP-glucose (glycone substrate for GtfE) with k(cat)/K(m) of 162.8 +/- 0.1 mM(-1) min(-1). Moreover, HMT31 showed improved substrate specificity toward seven more NDP-sugars. This study presents a domain swapping method as a potential means to glycorandomization toward various syntheses of 2-DOS-based aminoglycoside derivatives.     

Metabolic and functional diversity of saponins,biosynthetic intermediates and semi-synthetic derivatives

Abstract

Saponins are widely distributed plant natural products with vast structural and functional diversity. They are typically composed of a hydrophobic aglycone, which is extensively decorated with functional groups prior to the addition of hydrophilic sugar moieties, to result in surface-active amphipathic compounds. The saponins are broadly classified as triterpenoids, steroids or steroidal glycoalkaloids, based on the aglycone structure from which they are derived. The saponins and their biosynthetic intermediates display a variety of biological activities of interest to the pharmaceutical, cosmetic and food sectors. Although their relevance in industrial applications has long been recognized, their role in plants is underexplored. Recent research on modulating native pathway flux in saponin biosynthesis has demonstrated the roles of saponins and their biosynthetic intermediates in plant growth and development. Here, we review the literature on the effects of these molecules on plant physiology, which collectively implicate them in plant primary processes. The industrial uses and potential of saponins are discussed with respect to structure and activity, highlighting the undoubted value of these molecules as therapeutics.     

Engineered biosynthesis of glycosylated derivatives of narbomycin and evaluation of their antibacterial activities

A 14-membered macrolide antibiotic narbomycin produced from Streptomyces venezuelae ATCC 15439 is composed of polyketide macrolactone ring and D-desosamine as a deoxysugar moiety, which acts as an important determinant of its antibacterial activity. In order to generate diverse glycosylated derivatives of narbomycin, expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into S. venezuelae YJ003 mutant strain bearing a deletion of thymidine-5'-diphospho-D-desosamine biosynthetic gene cluster. The resulting recombinants of S. venezuelae produced a range of new analogs of narbomycin, which possess unnatural sugar moieties instead of native deoxysugar D-desosamine. The structures of narbomycin derivatives were determined through nuclear magnetic resonance spectroscopy and mass spectrometry analyses and their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with L-rhamnose or 3-O-demethyl-D-chalcose was demonstrated to exhibit greater antibacterial activity than narbomycin and the clinically relevant erythromycin. This work provides new insight into the functions of deoxysugar biosynthetic enzymes and structure-activity relationships of the sugar moieties attached to the macrolides and demonstrate the potential of combinatorial biosynthesis for the generation of new macrolides carrying diverse sugars with increased antibacterial activities.     

Superoxide radical- and peroxynitrite-scavenging activity of anthocyanins; structure-activity relationship and their synergism

Antioxidant activities of 15 purified bilberry anthocyanins together with pelargonidin 3-O-beta-D-glucopyranoside and 4'-O-methyl delphinidin 3-O-beta-D-glucopyranoside (MDp 3-glc), the major metabolite of delphinidin 3-O-beta-D-glucopyranoside (Dp 3-glc), were evaluated in order to study the structure-antioxidant activity relationship and any synergism among them in the mixture. Both aglycone structure and the attached sugar moiety affected the O*2- and ONOO- -scavenging activities, although the effect of the attached sugar moiety was smaller than that of the aglycone structure. The potency of activity toward the superoxide radical was in the following order: delphinidin > petunidin > malvidin =approximately cyanidin>(+)-catechin > peonidin > pelargonidin. The activity toward ONOO- was: delphinidin > cyanidin =approximately petunidin > malvidin =approximately (+)-catechin > peonidin > pelargonidin. It was confirmed that methylation of 4'-OH markedly reduced the antioxidant activity of anthocyanin. Further, it was revealed that synergism occurred in both - and ONOO- -scavenging activities among the anthocyanins in the mixture.     

Carboxyl-terminal domain characterization of polyene-specific P450 hydroxylase in <Emphasis Type="Italic">Pseudonocardia autotrophica</Emphasis>

A polyene compound NPP identified in Pseudonocardia autotrophica was shown to contain an aglycone identical to nystatin, but to harbor a unique disaccharide moiety that led to higher solubility and reduced hemolytic activity. Recently, it was revealed that the final step of NPP (nystatin-like polyene) biosynthesis is C10 regio-specific hydroxylation by the cytochrome P450 hydroxylase (CYP) NppL (Kim et al. [7]). Through mutation and cross-complementation, here we found that NppL preferred a polyene substrate containing a disaccharide moiety for C10 hydroxylation, while its orthologue NysL involved in nystatin biosynthesis showed no substrate preference toward mono- and disaccharide moieties, suggesting that two homologous polyene CYPs, NppL and NysL might possess a unique domain recognizing a sugar moiety. Two hybrid NppL constructs containing the C-terminal domain of NysL exhibited no substrate preference toward 10-deoxy NPP and 10-deoxy nystatin-like NysL, implying that the C-terminal domain plays a major role in differentiating the sugar moiety responsible for substrate specificity. Further C-terminal domain dissection of NppL revealed that the last fifty amino acids play a critical role in determining substrate specificity of polyene-specific hydroxylation, setting the stage for the biotechnological application of hydroxyl diversification for novel polyene biosynthesis in actinomycetes.     

Structure of a safflower steroid cellobioside

Structural identification of a steroid diglucoside from Carthamus tinctorius whose aglycone is 15α-20β-dihydroxy-Δ⁴-pregnen-3-one has been completed. We have analyzed the sugar moiety of the glycoside and found it to be cellobiose, β-linked to C-20 of the aglycone.     

Digitalis receptor sugar binding site characteristics: A model based upon studies of Na+, K+-ATPase preparations with differing digitalis sensitivities

The structure-activity relationships of the genin moieties of digitalis glycosides are commonly elucidated by determining the inhibitory potency of a variety of genins toward the plasma membrane Na⁺, K⁺-ATPase; qualitatively these relationships appear to be fairly independent of the specific Na⁺, K⁺-ATPase preparation utilized for the analysis. To determine whether this is the case with regard to the sugar moieties of glycosides, the inhibitory effects of 12 monoglycosides of digitoxigenin toward four Na⁺, K⁺-ATPase preparations of different origin were measured. It was found that while recognition of the major structural determinants of sugar activity appeared to be independent of enzyme source, recognition of the minor structural determinants of activity showed some source dependence. It was also observed that the intrinsic sensitivity to sugar potentiation may be source dependent and unrelated to intrinsic sensitivity to inhibition by digitoxigenin. These observations are compatible with a model of the Na⁺, K⁺-ATPase sugar binding site(s) in which intrinsic sensitivity to sugar attachment as well as recognition characteristics (for sugar structural features) both determine the extent to which a sugar moiety may contribute to the activity of monoglycosides. Further, in these studies one of the Na⁺, K⁺-ATPase preparations employed was obtained from rat brain, a tissue known to contain a mixture of ouabain sensitive and insensitive isoforms. We have observed that the rigorous purification techniques employed appear to have selectively removed from or denatured the less ouabain sensitive al isoform found in this enzyme preparation.     

Iridoid allosides from Viburnum opulus

Arbutin and four novel iridoid glycoside esters, named opulus iridoids I–IV, have been isolated from foliage of Viburnum opulus (Caprifoliaceae). Each opulus iridoid constitutes an inseparable mixture of two compounds, differing by containing either 2-methyl- or 3-methylbutyric acid in ester linkage at the 1-OH-group in an iridoid glycoside. In all glycosides 2′,3′-di-O-acetyl-β-D-allopyranose is linked through a glycosidic bond to C-11 in the iridoid aglycone. The opulus iridoids differ by the degree of acetylation of the aglycone and by the attachment, in III and IV, of a β-D-xylopyranosyl group at C-4 of the allose moiety. The structures have been elucidated by ¹H and ¹³C-NMR spectroscopy and by cleavage of the glycosidic linkage with boron trifluoride etherate in acetic anhydride, yielding the acetates of the cyclized aglycone and of the appropriate mono- or disaccharide. This is the second report of an iridoid attached to a sugar other than glucose and the second time allose has been encountered in higher plants. The systematic position of Viburnum is briefly discussed.     

Post-PKS Tailoring Steps of a Disaccharide-Containing Polyene NPP in Pseudonocardia autotrophica

A novel polyene compound NPP identified in a rare actinomycetes, Pseudonocardia autotrophica KCTC9441, was shown to contain an aglycone identical to nystatin but to harbor a unique di-sugar moiety, mycosaminyl-(α1-4)-N-acetyl-glucosamine, which led to higher solubility and reduced hemolytic activity. Although the nppDI was proved to be responsible for the transfer of first polyene sugar, mycosamine in NPP biosynthesis, the gene responsible for the second sugar extending glycosyltransferase (GT) as well as NPP post-PKS tailoring mechanism remained unknown. Here, we identified a NPP-specific second sugar extending GT gene named nppY, located at the edge of the NPP biosynthetic gene cluster. Targeted nppY gene deletion and its complementation proved that nppY is indeed responsible for the transfer of second sugar, N-acetyl-glucosamine in NPP biosynthesis. Site-directed mutagenesis on nppY also revealed several amino acid residues critical for NppY GT function. Moreover, a combination of deletions and complementations of two GT genes (nppDI and nppY) and one P450 hydroxylase gene (nppL) involved in the NPP post-PKS biosynthesis revealed that NPP aglycone is sequentially modified by the two different GTs encoded by nppDI and nppY, respectively, followed by the nppL-driven regio-specific hydroxylation at the NPP C10 position. These results set the stage for the biotechnological application of sugar diversification for the biosynthesis of novel polyene compounds in actinomycetes.     

Constituents from the bark of Tabebuia impetiginosa

The bark of Tabebuia impetiginosa afforded nineteen glycosides, consisting of four iridoid glycosides, two lignan glycosides, two isocoumarin glycosides, three phenylethanoid glycosides and eight phenolic glycosides. Their structures were determined using both spectroscopic and chemical methods. Iridoid glycosides, phenylethanoid glycosides and lignan glycosides had ajugol, osmanthuside H and secoisolariciresinol 4-O-beta-D-glucopyranoside as their structural elements, respectively, whereas the aglycone moieties of the isocoumarin glycosides were considered to be (-)-6-hydroxymellein. Phenolic glycosides had 4-methoxyphenol, 2,4-dimethoxyphenol, 3,4-dimethoxyphenol, 3,4,5-trimethoxyphenol and vanillyl 4-hydroxybenzoate as each aglycone moiety. Additionally, the sugar chains of these isocoumarin glycosides and phenolic glycosides were concluded to be beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside as well as those of osmanthuside H and above phenylethanoid glycosides.     

Glycosylation of Phenolic Compounds by the Site-Mutated β-Galactosidase from Lactobacillus bulgaricus L3

β-Galactosidases can transfer the galactosyl from lactose or galactoside donors to various acceptors and thus are especially useful for the synthesis of important glycosides. However, these enzymes have limitations in the glycosylation of phenolic compounds that have many physiological functions. In this work, the β-galactosidase from Lactobacillus bulgaricus L3 was subjected to site-saturation mutagenesis at the W980 residue. The recombinant pET-21b plasmid carrying the enzyme gene was used as the template for mutation. The mutant plasmids were transformed into Escherichia coli cells for screening. One recombinant mutant, W980F, exhibited increased yield of glycoside when using hydroquinone as the screening acceptor. The enzyme was purified and the effects of the mutation on enzyme properties were determined in detail. It showed improved transglycosylation activity on novel phenolic acceptors besides hydroquinone. The yields of the glycosides produced from phenol, hydroquinone, and catechol were increased by 7.6% to 53.1%. Moreover, it generated 32.3% glycosides from the pyrogallol that could not be glycosylated by the wild-type enzyme. Chemical structures of these glycoside products were further determined by MS and NMR analysis. Thus, a series of novel phenolic galactosides were achieved by β-galactosidase for the first time. This was a breakthrough in the enzymatic galactosylation of the challenging phenolic compounds of great values.     

Seven new steroidal glycosides from the roots of Cynanchum forrestii

Seven new steroidal glycosides, cynaforrosides K (1), L (2), M (3), N (4), and Q (7), based on a 13,14:14,15-disecopregnane-type aglycone, and cynaforrosides O (5) and P (6) with a 14,15-secopregnane-type aglycone, were isolated from the 95% ethanol extract of the roots of Cynanchum forrestii Schlechter. The structures of new compounds were determined on the basis of spectroscopic and chemical evidence. The sugar units of cynaforrosides K-P contained two moieties of glucoses with the mode of 1-->4 linkage and those of cynaforrosides K-O contained six moieties of sugars.