Glucosylation of benzyl alcohols by the cultured suspension cells of Nicotiana tabacum and Catharanthus roseus

The cultured cells of Nicotiana tabacum (white cells) converted regioselectively exogenous 2-, 3-, and 4-hydroxybenzyl alcohols into corresponding hydroxybenzyl-β-d-glucopyranoside. (RS)-1-Phenylethanol having chiral center in its substituent was also glucosylated to give 1-phenylethyl-β-d-glucopyranoside by the cultured cells of N. tabacum (white and green cells) and Catharanthus roseus. The glucosylation with the green cells of N. tabacum occurred enantioselectively to give the glucoside of (S)-alcohol preferentially, while the glucosylation with the white cells of N. tabacum and the C. roseus cells gave preferentially the glucoside of (R)-alcohol.     

Factors affecting the glucosylation capacity of cell cultures of Datura innoxia and Scopolia carniolica for monophenolic compounds

Suspension grown cells of Datura innoxia and Scopolia carniolica were tested for their glucosylation capacity and some factors affecting the efficiency of the reaction were studied.Cells at the end of the exponential growth phase showed a high glucosylation capacity. Light conditions had little effect on the bioconversion reaction. For the substrates hydroquinone and p-hydroxybenzoic acid the bioconversions were concentration-dependent. Permeabilization with propanol diminished the bioconversion capacity. Depending on the substrate used, relatively large amounts of substrate and product could not be recovered. Tannic acid could partly prevent decomposition of the compounds. The bioconversion capacity of cultures with a low glucosylation capacity could be enhanced by addition of uridine diphosphate-glucose, indicating that the sugar donor is a critical factor. From six substrates the natural compounds hydroquinone, p-hydroxybenzoic acid and vanillin were glucosylated. No glucosides were detected from tyramin and two synthetic aminotetralines.Abbreviations 5HAT 5-hydroxyaminotetralin - NO437 2-(N-propyl-N-2-thienylamino)-5-hydroxytetraline - pHBA p-hydroxybenzoic acid - UDP-glucose uridine diphosphate-glucose     

The Differentiation of Pigmentation in Flower Parts, IV. Flavonoid Elaborating Enzymes From Petals of Impatiens balsamina s

Extracts of the flower petals of Impatiens balsamina L. contain enzymes which catalyze the glycosylation of phenolic compounds. Enzymes have been extracted which glycosylate hydroquinone to arbutin and at least 3 different flavonols to the 3-monoglucoside. The hydroquinone glucosylating enzyme is similar to enzymes previously described except that it requires an unidentified low molecular weight cofactor. The glucosylation of flavonols follows normal enzyme kinetics; it requires a nucleotide diphosphate glucose donor for activity, and is made more evident by the presence of glucono-1:5-lactone, an inhibitor of endogenous glucosidases. It is suggested that the flavonol glucosylating enzyme acts naturally to glucosylate a precursor of both flavonols and anthocyanins to the 3-monoglucoside. The only elaboration of an anthocyanin observed with petal extracts was an acylation of pelargonidin-3-monoglucoside.     

Pathway for Biodegradation of p-Nitrophenol in a Moraxella sp

A Moraxella strain grew on p-nitrophenol with stoichiometric release of nitrite. During induction of the enzymes for growth on p-nitrophenol, traces of hydroquinone accumulated in the medium. In the presence of 2,2′-dipyridyl, p-nitrophenol was converted stoichiometrically to hydroquinone. Particulate enzymes catalyzed the conversion of p-nitrophenol to hydroquinone in the presence of NADPH and oxygen. Soluble enzymes catalyzed the conversion of hydroquinone to γ-hydroxymuconic semialdehyde, which was identified by high-performance liquid chromatography (HPLC)-mass spectroscopy. Upon addition of catalytic amounts of NAD⁺, γ-hydroxymuconic semialdehyde was converted to β-ketoadipic acid. In the presence of pyruvate and lactic dehydrogenase, substrate amounts of NAD were required and γ-hydroxymuconic semialdehyde was converted to maleylacetic acid, which was identified by HPLC-mass spectroscopy. Similar results were obtained when the reaction was carried out in the presence of potassium ferricyanide. Extracts prepared from p-nitrophenol-growth cells also contained an enzyme that catalyzed the oxidation of 1,2,4-benzenetriol to maleylacetic acid. The enzyme responsible for the oxidation of 1,2,4-benzenetriol was separated from the enzyme responsible for hydroquinone oxidation by DEAE-cellulose chromatography. The results indicate that the pathway for biodegradation of p-nitrophenol involves the initial removal of the nitro group as nitrite and formation of hydroquinone. 1,4-Benzoquinone, a likely intermediate in the initial reaction, was not detected. Hydroquinone is converted to β-ketoadipic acid via γ-hydroxymuconic semialdehyde and maleylacetic acid.     

Enzymatic synthesis of α-glucosyl-timosaponin BII catalyzed by the extremely thermophilic enzyme: Toruzyme 3.0L

Timosaponin BII (BII), a steroidal saponin showing potential anti-dementia activity, was converted into its glucosylation derivatives by Toruzyme 3.0L. Nine products with different degrees of glucosylation were purified and their structures were elucidated on the basis of ¹³C NMR, HR-ESI-MS, and FAB-MS spectra data. The active enzyme in Toruzyme 3.0L was purified to electrophoretic homogeneity by tracking BII-glycosylase activity and was identified as Cyclodextrin-glycosyltransferase (CGTase, EC 2.4.1.19) by ESI-Q-TOF MS/MS. In this work, we found that the active enzyme catalyzed the synthesis of alpha-(1→4)-linked glucosyl-BII when dextrin instead of an expensive activated sugar was used as the donor and showed a high thermal tolerance with the most favorable enzymatic activity at 100 °C. In addition, we also found that the α-amylases and CGTase, that is, GH13 family enzymes, all exhibited similar activities, which were able to catalyze glucosylation in steroidal saponins. But other kinds of amylases, such as γ-amylase (GH15 family), had no such activity under the same reaction conditions.     

High cell density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.     

HPLC-DAD analysis of arbutin produced from hydroquinone in a biotransformation process in Origanum majorana L. shoot culture

The hydroquinone glucoside arbutin is a plant derived compound medically applied due to its uroantiseptic activity. It also has skin whitening properties and thus is widely used in dermatology and cosmetology. Origanum majorana L. (Lamiaceae) is known to produce arbutin, however the content of the compound in cultivated plants is very variable and low. Since plant cell and tissue cultures are capable to perform specific biotransformation reactions including glucosylation, this investigation targeted the formation of arbutin from hydroquinone in agitated O. majorana shoot cultures. For this purpose different doses of hydroquinone (96, 144, 192, 288 and 384 mg/L of medium) were added to the culture flasks in one, two or three portions. Arbutin was qualitatively and quantitatively determined in methanol extracts from dry biomass and lyophilized media using HPLC-DAD. Cells of O. majorana shoot cultures efficiently converted hydroquinone into arbutin. The product was accumulated in the biomass and was not observed (or in trace amounts) in the medium samples. Different doses as well as portioning of the precursor had a significant impact on the biotransformation process. Arbutin accumulation increased from 0.23 ± 0.03 mg/g DW up to 52.6 ± 4.8 mg/g DW in the biomass. The highest product content was observed after the addition of 192 mg/L hydroquinone in three portions. The highest efficiency of the biotransformation process, i.e. 67.5 ± 5.2% was calculated for a dose of 96 mg/L precursor divided into three portions. After further optimization of the biotransformation process, O. majorana shoot cultures could serve as a rich source of arbutin.     

UDP-glucose:isoflavone 7-O-glucosyltransferase from roots of chick pea (Cicer arietinum L.)

A glucosyltransferase which catalyzes the glucosylation of isoflavones in position 7 using uridine diphosphate glucose as glucosyl donor has been purified about 120-fold from 4-day-old roots of chick pea (Cicer arietinum L.). The soluble enzyme showed a pH optimum of 8.5–9.0 and a molecular weight of 50,000. The K_m for uridine diphosphate glucose was 200 μm and for the isoflavones biochanin A and formononetin, 12 and 24 μm, respectively. While the aforementioned 4′-methoxy isoflavones were the best substrates, the 4′-hydroxy isoflavones genistein and daidzein were poor substrates. The enzyme was unable to catalyze the glucosylation of hydroxy substitutes isoflavanones, flavones, flavanones, flavonols, coumarins, cinnamic acids, and benzoic acids.     

Glucosylation of esculetin by plant cell suspension cultures

Cell suspension cultures of Lithospermum erythrorhizon, Gardenia jasminoides and Nicotiana tabacum were capable of glucosylating esculetin to esculin (7-hydroxycoumarin-6-O-β-D-glucoside). Especially, a culture strain of Lithospermum erythrorhizon was superior in the esculetin glucosylating capability; 40 to 50% of esculetin administered to the culture medium at early stationary growth stage was converted into esculin within 24 h. The rate of glucosylation was also dependent on the growth stage and the medium composition especially growth hormones and sugar.     

Effects of Sugars on the Glucosylation of Exogenous Hydroquinone by Catharanthus roseus Cells in Suspension Culture

The effects of sugars on the glucosylation of exogenous hydroquinone(HQ) was investigated by supplying individual sugars simultaneouslywith HQ to a suspension culture of Catharanthus roseus cells.The production of arbutin was enhanced as much as 2- to 3-foldby sucrose or glucose at concentrations of up to 6%, with theenhancement being directly dependent on the concentration ofthe sugar. The exogenously added sugar was not metabolized andremained unchanged. Sorbitol also promoted the production ofarbutin in a similar manner. Sucrose improved the viability of cells and, in cultures suppliedwith sucrose and HQ, the activity of UDP-glucose: hydroquinoneglucosyltransferase increased over a much longer period of timethan that in control cultures supplemented with HQ only. (Received December 11, 1989; Accepted March 26, 1990)     

Glucosylation of Steviol and Steviol-Glucosides in Extracts from Stevia rebaudiana Bertoni

To evaluate and characterize stevioside biosynthetic pathway in Stevia rebaudiana Bertoni cv Houten, two enzyme fractions that catalyze glucosylation of steviol (ent-13-hydroxy kaur-16-en-19-oic acid) and steviol-glucosides (steviol-13-O-glucopyranoside, steviolbioside and stevioside), utilizing UDP-glucose as the glucose donor, were prepared from the soluble extracts of S. rebaudiana leaves. Enzyme fraction I, passed through DEAE-Toyopearl equilibrated with 50 millimolar K-phosphate pH 7.5, catalyzed the glucosylation to steviol and 19-O-methylsteviol, but not to iso-steviol and 13-O-methylsteviol, indicating that 13-hydroxyl group of the steviol skeleton is glucosylated first from UDP-glucose to produce steviol-13-O-glucopyranoside. Enzyme fraction II, eluted from the DEAE-Toyopearl column with 0.15 molar KCI, catalyzed the glucose transfer from UDP-glucose to steviol-13-O-glucopyranoside, steviolbioside and stevioside, but not to rubusoside (13, 19-di-O-glucopyranoside) and rebaudioside A. The reaction products glucosylated from steviol-13-O-glucopyranoside, steviolbioside and stevioside were identified to be steviolbioside, stevioside and rebaudioside A, respectively. These results indicate that in the steviol-glucoside biosynthetic pathway, steviol-13-O-glucopyranoside produced from the steviol glucosylation is successively glucosylated to steviolbioside, then to stevioside producing rebaudioside A.     

Effect of substituent groups on the glucosyl conjugation of xenobiotic phenols by cultured cells of Gardenia jasminoides

Phenol, nitrophenols and hydroxyphenols added exogenously to Gardenia jasminoides Ellis cell suspension cultures were conjugated to form the corresponding β,d-monoglucosides. The reaction proceeded linearly after the addition of the substrates, indicating participation of constitutive glucosyltransferases in the cultured cells. Nitro substitution, especially in the ortho-position, enhanced the velocity of glucosyl conjugation. Ortho-hydroxyphenol (catechol) was also glucosylated with a higher velocity than phenol.These results suggest that the acidity of a phenolic hydroxyl group and position of a substituent can play important roles in the glucosylation of phenolic compounds by cultured cells of G. jasminoides.     

Quantification of small GTPase glucosylation by clostridial glucosylating toxins using multiplexed MRM analysis

Large clostridial toxins mono‐O‐glucosylate small GTPases of the Rho and Ras subfamily. As a result of glucosylation, the GTPases are inhibited and thereby corresponding downstream signaling pathways are disturbed. Current methods for quantifying the extent of glucosylation include sequential [¹⁴C]glucosylation, sequential [³²P]ADP‐ribosylation, and Western Blot detection of nonglucosylated GTPases, with neither method allowing the quantification of the extent of glucosylation of an individual GTPase. Here, we describe a novel MS‐based multiplexed MRM assay to specifically quantify the glucosylation degree of small GTPases. This targeted proteomics approach achieves a high selectivity and reproducibility, which allows determination of the in vivo substrate pattern of glucosylating toxins. As proof of principle, GTPase glucosylation was analyzed in CaCo‐2 cells treated with TcdA, and glucosylation kinetics were determined for RhoA/B, RhoC, RhoG, Ral, Rap1, Rap2, (H/K/N)Ras, and R‐Ras2.     

Enzymatic synthesis of apigenin glucosides by glucosyltransferase (YjiC) from Bacillus licheniformis DSM 13

Apigenin, a member of the flavone subclass of flavonoids, has long been considered to have various biological activities. Its glucosides, in particular, have been reported to have higher water solubility, increased chemical stability, and enhanced biological activities. Here, the synthesis of apigenin glucosides by the in vitro glucosylation reaction was successfully performed using a UDP-glucosyltransferase YjiC, from Bacillus licheniformis DSM 13. The glucosylation has been confirmed at the phenolic groups of C-4′ and C-7 positions ensuing apigenin 4′-O-glucoside, apigenin 7-O-glucoside and apigenin 4′,7-O-diglucoside as the products leaving the C-5 position unglucosylated. The position of glucosylation and the chemical structures of glucosides were elucidated by liquid chromatography/mass spectroscopy and nuclear magnetic resonance spectroscopy. The parameters such as pH, UDP glucose concentration and time of incubation were also analyzed during this study.     

Actin Re-Organization Induced by Chlamydia trachomatis Serovar D - Evidence for a Critical Role of the Effector Protein CT166 Targeting Rac

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs). CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB) or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut) exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1) from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation. Finally, chlamydial uptake was impaired in CT166 over-expressing cells. Our data strongly suggest CT166''s participation as an effector protein during host-cell entry, ensuring a balanced uptake into host-cells by interfering with Rac-dependent cytoskeletal changes.     

Substrate Utilization by Suspension Cultures and Somatic Embryos of Daucus carota L. Measured by C NMR

The uptake and utilization of sucrose by embryogenic suspension cultures of carrot (Daucus carota L.) growing in the presence of 2,4-D and by somatic embryos derived from these cultures was monitored using ¹³C nuclear magnetic resonance. The exogeneously supplied sucrose was completely hydrolyzed before cell entry; glucose was taken up preferentially when the cells were cultured in the presence of 2,4-D, while glucose and fructose were utilized at similar rates by somatic embryos in the absence of 2,4-D. Both suspension cells and somatic embryos accumulated high intracellular levels predominantly of glucose and sucrose, the latter being resynthesized intracellularly from the constitutive hexoses. Initially, fructose was converted mainly into glucose and sucrose rather than being catabolized directly through glycolysis or the pentose phosphate pathway. Carbohydrate supply that exceeded cellular demand resulted in intracellular accumulation of mono- or disaccharides. The capacity of cultured carrot cells to produce somatic embryos appeared to be positively correlated with high intracellular levels of glucose.     

Carbohydrate metabolism of hepatocytes from starved Japanese quail

Hepatocytes were isolated from livers of mature male and female starved Japanese quail (Coturnix coturnix japonica). The hepatocytes take up lactate and dihydroxyacetone extensively, and have a very high rate of glucose synthesis from these substrates. Fructose uptake and incorporation into glucose is much less. Pyruvate and alanine are taken up extensively, but form little glucose. There is negligible lipogenesis in cells of starved quail. Alanine increases up to 10-fold incorporation of ³HOH and ¹⁴C from several substrates into fatty acids, but it remains insignificant as compared to lipogenesis by cells of fed quail. There is little utilization of glucose, even in the presence of alanine, in marked contrast to hepatocytes from fed quail. However, glucose is phosphorylated at high rates, but most of the glucose 6-phosphate is recycled to glucose. There is a marked difference in the metabolism of polyols between the sexes. Glycerol, xylitol, and sorbitol are converted nearly quantitatively into glucose by hepatocytes of starved female quail. In cells of starved males, the uptake of polyols is higher, but conversion to glucose less efficient. In cells of starved male quail, alanine markedly stimulates the uptake of glycerol and xylitol and their conversion to glucose, but has no effect on sorbitol metabolism. In cells of female quail, alanine is without a significant effect on polyol metabolism.     

Glucosylation of Lipopolysaccharide in Salmonella: Mutants Negative for O Antigen Factor 122

In salmonella O group B, the O antigen factor 12₂ is created by glucosylation at C4 of the galactose units of the O side chains; phage-determined glucosylation at C6 of these galactose units yields factor 1. 12₂-negative mutants were isolated from 12₂⁺ (stable) parents (genetically oafR^+st). All 20 mutants studied were stable in respect of their 12₂ character. In eight of them, lysogenization by phage P22 resulted in the appearance of the 12₂ factor—these were interpreted to be defective in the enzyme(s) needed to synthesize the glucose-lipid intermediate that participates in both 12₂- and 1-specific glucosylation; the corresponding cistron was termed oafE. This “complementation” assay also demonstrated that the P22 genome can determine the synthesis of this glucose-lipid intermediate. The 12₂ character in the oafE⁻ mutants became variable after P22 lysogenization, corresponding to factor 1 variation normally determined by this phage. In the remaining 12 mutants, P22 caused the appearance of a variable factor 1, but not of 12₂. The lesion in all the 12₂ mutants was mapped close to the gene purE at min 19 of the Salmonella map. This is the same area where the locus oafR that controls the variation (stable or variable, + or −) of 12₂ had been previously mapped. The results suggest the presence of a 12₂ glucosylation operon in this region.     

Stability of the Plasma Membrane in Saccharomyces rouxii and Its Relationship to Glucose Tolerance

The stability of spheroplasts from the osmotrophic yeast Saccharomyces rouxii was studied in buffered solutions of mannitol and glucose. The plasma membranes from cells grown in high glucose concentrations were more stable to osmotic lysis than were membranes from cells grown in lower glucose concentrations. Mannitol was a better osmotic stabilizer than glucose, except when the cells were grown in a high glucose concentration. Spheroplasts from a glucose tolerant-deficient mutant were much less stable than the corresponding spheroplasts from the parent strain, especially when suspended in glucose solutions. These results suggest an involvement of the plasma membrane in the glucose-tolerant mechanism of S. rouxii.     

Glycation of Human Cortical and Cancellous Bone Captures Differences in the Formation of Maillard Reaction Products between Glucose and Ribose

To better understand some aspects of bone matrix glycation, we used an in vitro glycation approach. Within two weeks, our glycation procedures led to the formation of advanced glycation end products (AGEs) at the levels that corresponded to approx. 25–30 years of the natural in vivo glycation. Cortical and cancellous bones from human tibias were glycated in vitro using either glucose (glucosylation) or ribose (ribosylation). Both glucosylation and ribosylation led to the formation of higher levels of AGEs and pentosidine (PEN) in cancellous than cortical bone dissected from all tested donors (young, middle-age and elderly men and women). More efficient glycation of bone matrix proteins in cancellous bone most likely depended on the higher porosity of this tissue, which facilitated better accessibility of the sugars to the matrix proteins. Notably, glycation of cortical bone from older donors led to much higher AGEs levels as compared to young donors. Such efficient in vitro glycation of older cortical bone could result from aging-related increase in porosity caused by the loss of mineral content. In addition, more pronounced glycation in vivo would be driven by elevated oxidation processes. Interestingly, the levels of PEN formation differed pronouncedly between glucosylation and ribosylation. Ribosylation generated very high levels of PEN (approx. 6- vs. 2.5-fold higher PEN level than in glucosylated samples). Kinetic studies of AGEs and PEN formation in human cortical and cancellous bone matrix confirmed higher accumulation of fluorescent crosslinks for ribosylation. Our results suggest that in vitro glycation of bone using glucose leads to the formation of lower levels of AGEs including PEN, whereas ribosylation appears to support a pathway toward PEN formation. Our studies may help to understand differences in the progression of bone pathologies related to protein glycation by different sugars, and raise awareness for excessive sugar supplementation in food and drinks.