Glycosylation of Ganoderic Acid G by Bacillus Glycosyltransferases.
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ABSTRACT:
SUBMITTER: Wu JY
PROVIDER: S-EPMC8468440 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bacillus subtilis ATCC (American type culture collection) 6633 was found to biotransform ganoderic acid A (GAA), which is a major lanostane triterpenoid from the medicinal fungus Ganoderma lucidum. Five glycosyltransferase family 1 (GT1) genes of this bacterium, including two uridine diphosphate-dependent glycosyltransferase (UGT) genes, BsUGT398 and BsUGT489, were cloned and overexpressed in Escherichia coli. Ultra-performance liquid chromatography confirmed the two purified UGT proteins biotransform ganoderic acid A into a metabolite, while the other three purified GT1 proteins cannot biotransform GAA. The optimal enzyme activities of BsUGT398 and BsUGT489 were at pH 8.0 with 10 mM of magnesium or calcium ion. In addition, no candidates showed biotransformation activity toward antcin K, which is a major ergostane triterpenoid from the fruiting bodies of Antrodia cinnamomea. One biotransformed metabolite from each BsUGT enzyme was then isolated with preparative high-performance liquid chromatography. The isolated metabolite from each BsUGT was identified as ganoderic acid A-15-O-β-glucoside by mass and nuclear magnetic resonance spectroscopy. The two BsUGTs in the present study are the first identified enzymes that catalyze the 15-O-glycosylation of triterpenoids.
| S-EPMC6275011 | biostudies-literature
Project description:Ganoderic acid A (GAA) is a bioactive triterpenoid isolated from the medicinal fungus Ganoderma lucidum. Our previous study showed that the Bacillus subtilis ATCC (American type culture collection) 6633 strain could biotransform GAA into compound (1), GAA-15-O-?-glucoside, and compound (2). Even though we identified two glycosyltransferases (GT) to catalyze the synthesis of GAA-15-O-?-glucoside, the chemical structure of compound (2) and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the same B. subtilis strain, for the biotransformation of GAA into compound (2) through acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a scaled-up production, compound (2) was successfully isolated using preparative high-performance liquid chromatography and identified to be a new triterpenoid glucoside (GAA-26-O-?-glucoside) by mass and nuclear magnetic resonance spectroscopy. The results of kinetic experiments showed that the turnover number (kcat) of BsGT110 toward GAA at pH 6 (kcat = 11.2 min-1) was 3-fold higher than that at pH 7 (kcat = 3.8 min-1), indicating that the glycosylation activity of BsGT110 toward GAA was more active at acidic pH 6. In short, we determined that BsGT110 is a unique GT that plays a role in the glycosylation of triterpenoid at the C-26 position under acidic conditions, but loses most of this activity under alkaline ones, suggesting that acidic solutions may enhance the catalytic activity of this and similar types of GTs toward triterpenoids.
| S-EPMC6804120 | biostudies-literature
Project description:Strain GA A07 was identified as an intestinal Bacillus bacterium of zebrafish, which has high efficiency to biotransform the triterpenoid, ganoderic acid A (GAA), into GAA-15-O-?-glucoside. To date, only two known enzymes (BsUGT398 and BsUGT489) of Bacillus subtilis ATCC 6633 strain can biotransform GAA. It is thus worthwhile to identify the responsible genes of strain GA A07 by whole genome sequencing. A complete genome of strain GA A07 was successfully assembled. A phylogenomic analysis revealed the species of the GA A07 strain to be Bacillus thuringiensis. Forty glycosyltransferase (GT) family genes were identified from the complete genome, among which three genes (FQZ25_16345, FQZ25_19840, and FQZ25_19010) were closely related to BsUGT398 and BsUGT489. Two of the three candidate genes, FQZ25_16345 and FQZ25_19010, were successfully cloned and expressed in a soluble form in Escherichia coli, and the corresponding proteins, BtGT_16345 and BtGT_19010, were purified for a biotransformation activity assay. An ultra-performance liquid chromatographic analysis further confirmed that only the purified BtGT_16345 had the key biotransformation activity of catalyzing GAA into GAA-15-O-?-glucoside. The suitable conditions for this enzyme activity were pH 7.5, 10 mM of magnesium ions, and 30 °C. In addition, BtGT_16345 showed glycosylation activity toward seven flavonoids (apigenein, quercetein, naringenein, resveratrol, genistein, daidzein, and 8-hydroxydaidzein) and two triterpenoids (GAA and antcin K). A kinetic study showed that the catalytic efficiency (kcat/KM) of BtGT_16345 was not significantly different compared with either BsUGT398 or BsUGT489. In short, this study identified BtGT_16345 from B. thuringiensis GA A07 is the catalytic enzyme responsible for the 15-O-glycosylation of GAA and it was also regioselective toward triterpenoid substrates.
| S-EPMC6829469 | biostudies-literature
Project description:Glycosylation of natural products can influence their pharmacological properties, and efficient glycosyltransferases (GTs) are critical for this purpose. The polyketide epothilones are potent anti-tumour compounds, and YjiC is the only reported GT for the glycosylation of epothilone. In this study, we phylogenetically analysed 8261 GTs deposited in CAZy database and revealed that YjiC locates in a subbranch of the Macrolide I group, forming the YjiC-subbranch with 160 GT sequences. We demonstrated that the YjiC-subbranch GTs are normally efficient in epothilone glycosylation, but some showed low glycosylation activities. Sequence alignment of YjiC-subbranch showed that the 66th and 77th amino acid residues, which were close to the catalytic cavity in molecular docking model, were conserved in five high-active GTs (Q66 and P77) but changed in two low-efficient GTs. Site-directed residues swapping at the two positions in the two low-active GTs (BssGT and BamGT) and the high-active GT BsGT-1 demonstrated that the two amino acid residues played an important role in the catalytic efficiency of epothilone glycosylation. This study highlights that the potent GTs for appointed compounds are phylogenetically grouped with conserved residues for the catalytic efficiency.
| S-EPMC6559208 | biostudies-literature
Project description:Functional bacteria that could biotransform triterpenoids may exist in the diverse microflora of fish intestines. Ganoderic acid A (GAA) is a major triterpenoid from the medicinal fungus Ganoderma lucidum. In studying the microbial biotransformation of GAA, dozens of intestinal bacteria were isolated from the excreta of zebrafish. The bacteria's ability to catalyze GAA were determined using ultra-performance liquid chromatography analysis. One positive strain, GA A07, was selected for functional studies. GA A07 was confirmed as Bacillus sp., based on the DNA sequences of the 16S rRNA gene. The biotransformed metabolite was purified with the preparative high-performance liquid chromatography method and identified as GAA-15-O-?-glucoside, based on the mass and nuclear magnetic resonance spectral data. The present study is the first to report the glycosylation of Ganoderma triterpenoids. Moreover, 15-O-glycosylation is a new microbial biotransformation of triterpenoids, and the biotransformed metabolite, GAA-15-O-?-glucoside, is a new compound.
| S-EPMC6225395 | biostudies-literature
Project description:Protein glycosylation is a common post-translational modification that influences the functions and properties of proteins. Despite advances in methods to produce defined glycoproteins by chemoenzymatic elaboration of monosaccharides, the understanding and engineering of glycoproteins remain challenging, in part, due to the difficulty of site-specifically controlling glycosylation at each of several positions within a protein. Here, we address this limitation by discovering and exploiting the unique, conditionally orthogonal peptide acceptor specificities of N-glycosyltransferases (NGTs). We used cell-free protein synthesis and mass spectrometry of self-assembled monolayers to rapidly screen 41 putative NGTs and rigorously characterize the unique acceptor sequence preferences of four NGT variants using 1254 acceptor peptides and 8306 reaction conditions. We then used the optimized NGT-acceptor sequence pairs to sequentially install monosaccharides at four sites within one target protein. This strategy to site-specifically control the installation of N-linked monosaccharides for elaboration to a variety of functional N-glycans overcomes a major limitation in synthesizing defined glycoproteins for research and therapeutic applications.
| S-EPMC7047269 | biostudies-literature
Project description:The process of glycosylation has been studied extensively in prokaryotes but many questions still remain unanswered. Glycosyltransferase is the enzyme which mediates glycosylation and has its preference for the target glycosylation sites as well as for the type of glycosylation i.e. N-linked and O-linked glycosylation. In this study we carried out the bioinformatics analysis of one of the key enzymes of pgl locus from Campylobacter jejuni, known as PglB, which is distributed widely in bacteria and AglB from archaea. Relatively little sequence similarity was observed in the archaeal AglB(s) as compared to those of the bacterial PglB(s). In addition we tried to the answer the question of as to why not all the sequins Asp-X-Ser/Thr have an equal opportunity to be glycosylated by looking at the influence of the neighboring amino acids but no significant conserved pattern of the flanking sites could be identified. The software tool was developed to predict the potential glycosylation sites in autotransporter protein, the virulence factors of gram negative bacteria, and our results revealed that the frequency of glycosylation sites was higher in adhesins (a subclass of autotransporters) relative to the other classes of autotransporters.
| S-EPMC3124799 | biostudies-literature
Project description:Ganoderic acid A (GAA), a representative active triterpenoid from Ganoderma lucidum, has been reported to exhibit antinociceptive, antioxidative, cytotoxic, hepatoprotective and anticancer activities. The present study aims (1) to identify GAA metabolites, in vivo by analyzing the bile, plasma and urine after intravenous administration to rats (20 mg/kg), and in vitro by incubating with rat liver microsomes (RLMs) and human liver microsomes (HLMs); (2) to investigate the metabolic kinetics of main GAA metabolites. Using HPLC-DAD-MS/MS techniques, a total of 37 metabolites were tentatively characterized from in vivo samples based on their fragmentation behaviors. The metabolites detected in in vitro samples were similar to those found in vivo. GAA underwent extensive phase I and II metabolism. The main metabolic soft spots of GAA were 3, 7, 11, 15, 23-carbonyl groups (or hydroxyl groups) and 12, 20, 28 (29)-carbon atoms. Ganoderic acid C2 (GAC2) and 7β,15-dihydroxy-3,11,23-trioxo-lanost-26-oic acid were two main reduction metabolites of GAA, and their kinetics followed classical hyperbolic kinetics. The specific isoenzyme responsible for the biotransformation of the two metabolites in RLMs and HLMs was CYP3A. This is the first report on the comprehensive metabolism of GAA, as well as the metabolic kinetics of its main metabolites.
| S-EPMC5339268 | biostudies-literature
Project description:Eukaryotic protein glycosylation is mediated by glycosyl- and oligosaccharyl-transferases. Here, we describe how African trypanosomes exhibit both evolutionary conservation and significant divergence compared with other eukaryotes in how they synthesise their glycoproteins. The kinetoplastid parasites have conserved components of the dolichol-cycle and oligosaccharyltransferases (OSTs) of protein N-glycosylation, and of glycosylphosphatidylinositol (GPI) anchor biosynthesis and transfer to protein. However, some components are missing, and they process and decorate their N-glycans and GPI anchors in unique ways. To do so, they appear to have evolved a distinct and functionally flexible glycosyltransferases (GT) family, the GT67 family, from an ancestral eukaryotic β3GT gene. The expansion and/or loss of GT67 genes appears to be dependent on parasite biology. Some appear to correlate with the obligate passage of parasites through an insect vector, suggesting they were acquired through GT67 gene expansion to assist insect vector (tsetse fly) colonisation. Others appear to have been lost in species that subsequently adopted contaminative transmission. We also highlight the recent discovery of a novel and essential GT11 family of kinetoplastid parasite fucosyltransferases that are uniquely localised to the mitochondria of Trypanosoma brucei and Leishmania major. The origins of these kinetoplastid FUT1 genes, and additional putative mitochondrial GT genes, are discussed.
| S-EPMC9472816 | biostudies-literature
Project description:Glycosyltransferases, usually residing within the intracellular secretory apparatus, also circulate in the blood. Many of these blood-borne glycosyltransferases are associated with pathological states, including malignancies and inflammatory conditions. Despite the potential for dynamic modifications of glycans on distal cell surfaces and in the extracellular milieu, the glycan-modifying activities present in systemic circulation have not been systematically examined. Here, we describe an evaluation of blood-borne sialyl-, galactosyl- and fucosyltransferase activities that act upon the four common terminal glycan precursor motifs, GlcNAc monomer, Gal(β3)GlcNAc, Gal(β4)GlcNAc and Gal(β3)GalNAc, to produce more complex glycan structures. Data from radioisotope assays and detailed product analysis by sequential tandem mass spectrometry show that blood has the capacity to generate many of the well-recognized and important glycan motifs, including the Lewis, sialyl-Lewis, H- and Sialyl-T antigens. While many of these glycosyltransferases are freely circulating in the plasma, human and mouse platelets are important carriers for others, including ST3Gal-1 and β4GalT. Platelets compartmentalize glycosyltransferases and release them upon activation. Human platelets are also carriers for large amounts of ST6Gal-1 and the α3-sialyl to Gal(β4)GlcNAc sialyltransferases, both of which are conspicuously absent in mouse platelets. This study highlights the capability of circulatory glycosyltransferases, which are dynamically controlled by platelet activation, to remodel cell surface glycans and alter cell behavior.
| S-EPMC5224594 | biostudies-literature