Flavanone-7-O-glucosyltransferase activity from Petunia hybrida

Citrus spp. are known for the accumulation of flavanone glycosides (e.g., naringin comprises up to 70% of the dry weight of very young grapefruit). In contrast, petunia utilizes relatively more naringenin for production of flavonol glycosides and anthocyanins. This investigation addressed whether or not petunia is capable of glucosylation of naringenin and if so, what are the characteristics of this flavanone glucosylating enzyme. Petunia leaf tissue contains some flavanone-7-O-glucosyltransferase (E.C. 2.4.1.185) activity, although at 90-fold lower levels than grapefruit leaves. This activity was partially purified 89-fold via ammonium sulfate fractionation followed by FPLC on Superose 12 and Mono Q yielding three chromatographically separate peaks of activity. The enzymes in the peak fractions glucosylated flavanone, flavonol, and flavone substrates. Enzymes in Mono Q peaks I and II were relatively more specific toward flavanone substrates and peak I was significantly more active. Enzyme activity was not effected by Ca2+, Mg2+, AMP, ADP, or ATP. The petunia enzyme was over 10,000 times more sensitive to UDP inhibition (Ki 0.89 microM) than the flavanone-specific 7GT in grapefruit. These and other results suggest that different flavanoid accumulation patterns in these two plants may be partially due to the different relative levels and biochemical properties of their flavanone glucosylating (7GT) enzymes.     

Modulation of activities of steryl glucoside hydrolase and UDPG: Sterol glucosyltransferase from Sinapis alba by detergents and lipids

Interactions of detergents and lipid compounds on the activity of delipidated preparations of UDPG: sterol glucosyltransferase and steryl β-d-glucoside hydrolase (SG hydrolase) isolated from white mustard seedlings were studied. It has been found that various lipids exert diverse effects on the activity of SG hydrolase. This activity was distinctly stimulated by several neutral, relatively unpolar compounds such as phytol, tripalmitoylglycerol, methyl stearate or cholesteryl acetate and, to a lesser extent, by free fatty acids. On the other hand a number of phospho- and glycolipids were inhibitory. A particularly strong inhibition was observed with charged, zwitterionic phospholipids such as PC, PE or their 2-lyso derivatives. These results point to the possibility of in vivo regulation of the membrane-bound SG hydrolase by its lipid microenvironment. In contrast to SG hydrolase no evidence was found for a clear-cut effect of lipids on the activity of UDPG: sterol glucosyltransferase even after a pretreatment of the enzyme preparation with phospholipase C.     

The estimation of phenylalanine ammonia-lyase (PAL)-activity in intact cells of higher plant tissue

From cell cultures of Haplopappus gracilis, an enzyme, catalyzing the glucosylation of cyanidin at the 3 position using uridine diphosphate-D-glucose (UDPG) as glucosyl-donor, has been isolated and purified 50-fold. The enzyme was not specific for cyanidin alone, but also glucosylated other anthocyanidins and flavonols in position 3. However, apigenin, luteolin, naringenin and dihydroquercetin were not glucosylated. The reaction has an optimum pH of approximately 8, and the apparent K _m values for UDPG and cyanidin were 0.5 and 0.33 mM respectively. The enzyme reaction is strongly inhibited by cyanidin (above 0.25 mM).     

Biosynthesis of cyanogenic glucosides: in vitro analysis of the glucosylation step

The last step in the biosynthesis of cyanogenic glucosides, the glucosylation of the cyanohydrin intermediate, has been investigated in detail using Triglochin maritima seedlings. The glucosyltransferase activity is not associated with membranes and appears to be a "soluble" enzyme. The cyanohydrin intermediate, which is formed by hydroxylation of 4-hydroxyphenylacetonitrile by a membrane-bound enzyme, is free to equilibrate in the presence of the glucosyltransferase and UDPG, because it can be trapped very efficiently. This indicates that this intermediate is not channeled (unlike some of the other intermediates), although it is probably the most labile of all of them. The glucosyltransferase of T. maritima responsible for the glucosylation of the cyanohydrin was separated from another glucosyltransferase, which used 4-hydroxybenzylalcohol as a substrate, and purified over 200-fold. It catalyzed the glucose transfer from UDPG to only 4-hydroxymandelonitrile and 3,4-dihydroxymandelonitrile, giving rise to the respective cyanogenic glucosides. Although the activities with these two substrates behaved differently in certain respects (e.g., extent of inactivation during purification and difference in activation by higher salt concentrations), most of the data acquired favor the view that only one enzyme in T. maritima is responsible for the glucosylation of both substrates.     

Naringenin inhibits phosphoinositide 3-kinase activity and glucose uptake in 3T3-L1 adipocytes

Previous studies have shown that flavonoids inhibit glucose uptake in cultured cells. In this report, we show that the grapefruit flavanone naringenin inhibited insulin-stimulated glucose uptake in 3T3-L1 adipocytes in a dose-dependent manner. Naringenin acts by inhibiting the activity of phosphoinositide 3-kinase (PI3K), a key regulator of insulin-induced GLUT4 translocation. Although naringenin did not alter the phosphotyrosine status of the insulin receptor, insulin receptor substrate proteins, or PI3K, it did inhibit the phosphorylation of the downstream signaling molecule Akt. In an in vitro kinase assay, naringenin inhibited PI3K activity. A physiologically attainable dose of 6 microM naringenin reduced insulin-stimulated glucose uptake by approximately 20%. This inhibitory effect remained 24h after the removal of naringenin from the culture medium. Collectively, our findings suggest that the regular consumption of naringenin in grapefruit may exacerbate insulin resistance in susceptible individuals via impaired glucose uptake in adipose tissue.     

Glucose and sulfate conjugation of phenolic compounds by the spiny lobster (Panulirus argus)

Previous work has shown that the hepatopancreas of the spiny lobster (Panulirus argus) contains a mixed-function oxidase system capable of catalyzing the monooxygenation of polycyclic aromatic hydrocarbons to highly toxic products similar to those formed by mammalian tissues. Studies were designed to determine the ability of the spiny lobster to conjugate the phenolic compounds 4-methylumbelliferone, p-nitrophenol, beta-naphthol, and 3-hydroxybenzo[a]pyrene with endogenous molecules. The hepatopancreas contained UDP-glucose (UDPG) dependent glucosyltransferase, while no activity was detected when UDP-glucuronic acid was used as the cosubstrate. Atypical Michaelis-Menten kinetics result with varying concentrations of UDPG, indicating that multiple forms of glucosyltransferase may exist in this organ. The activity was localized in the microsomal fraction, exhibited a pH optimum at 8.0-8.5, and a temperature optimum of 30 degrees C. Sulfate conjugation was found only in the cytosolic fraction of the antennal gland and used adenosine 3'-phosphate 5'-phosphosulfate (PAPS) as the sulfate donor (Km(apparent) = 9.0 +/- 4.9 microM). Hepatopancreas cytosol inhibited sulfotransferase activity. The pH optimum of antennal gland sulfotransferase was a function of the substrate and ranged from 5.5 to 7.4. Analysis of spiny lobster urine 24 hr following exposure to 3-hydroxybenzo[a]pyrene demonstrated the ability of the lobster to form both the sulfate and glucoside conjugate in vivo.     

Subcellular localization of udpg: Nuatigenin glucosyltransferase in oat leaves

Cell-free enzyme preparations from oat leaves effectively catalyse the conversion of both phytosterols and nuatigenin (a furostanol sapogenin) to the corresponding 3β-d-glucosides, with UDPG acting as a sugar donor. Subcellular fractionation has shown that UDPG: sterol glucosyltransferase activity is present almost exclusively in the membranous fraction (105 000 g pellet) while a large part (ca 70 %) of UDPG : nuatigenin glucosyltransferase activity occurs in the cytosol (105 000 g supernatant). The results obtained indicate clearly that oat leaves contain at least two UDPG-dependent glucosyltransferases catalysing glucosylation of 3β-hydroxysteroids which are localized in different cell compartments and exhibit different specifirity patterns.     

Occurrence and characterization of a UDP-glucose:hydroxamic acid glucosyltransferase isolated from wheat (Triticum aestivum) seedlings

Cyclic hydroxamic acid glucosides are present at high concentrations immediately after germination in wheat (Triticum aestivum L.). Changes in the activity of UDP-Glucose:cyclic hydroxamic acid glucosyltransferase (EC 2.4.1.-) in wheat were investigated using the cyclic hydroxamic acids 2.4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy derivative (DIMBOA) as sugar acceptors. Glucosyltransferase activity on both substrates was detected in dry seeds, with activity increasing after imbibition, peaking in shoots and roots 36-48 hours after imbibition and decreasing thereafter. The transience of glucosyltransferase activity was concurrent with the transient occurrence of the hydroxamic acid glucosides [Nakagawa E., Amano T., Hirai N., and Iwamura H. (1995) Phytochemistry 38, 1349-1354], suggesting that glucosyltransferases regulate the accumulation of hydroxamic acid glucosides in wheat seedlings. Two peaks in activity of UDP-Glucose:DIMBOA glucosyltransferase were detected using a Mono Q column, indicating the presence of at least two isozymes of this glucosyltransferase. The enzyme in the major peak was purified about 1500-fold and shown to be in a monomeric form with a molecular mass of 47 or 49 kDa. The enzyme reacted strongly with DIMBOA, less so with DIBOA. The enzyme of the minor peak on the Mono Q chromatogram, which was also a monomeric enzyme with a molecular mass of 47 kDa, showed similar substrate specificity to that of the major peak enzyme.     

Flavonol glucosyltransferase activity in Bronze embryos of Zea mays

The major UDPG: flavonol glucosyltransferase (UFGT) in maize is an enzyme of strict positional specificity known to be coded by the Bz locus. In bz mature endosperms, no UFGT can be detected. However, bz embryos possess a residual flavonol glucosyltransferase activity which is independent of Bz locus control. The products of this activity have been identified as the 3′-, 7- and 3-glucosides.     

Flavanone 3-hydroxylase expression in Citrus paradisi and Petunia hybrida seedlings

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.     

UDP-Glucose: Cyanidin 3-O-glucosyltransferase from red cabbage seedlings

An enzyme, catalysing the glucosylation of cyanidin at the 3-position using uridine diphosphate-D-glucose (UDPG) as glucosyl-donor, has been isolated and purified about 50-fold from young red cabbage (Brassica oleracea) seedlings. The pH optimum for this reaction was ca 8 and no additional cofactors were required. The reaction was inhibited by cyanidin (above 0.25 mM) and by very low concentrations of the reaction product cyanidin-3-glucoside (5 μM). The K_m values for UDPG and cyanidin were 0.51 and 0.4 mM respectively. In addition to cyanidin the enzyme could also glucosylate the following compounds at the 3-position: pelargonidin, peonidin, malvidin, kaempferol, quercetin, isorhamnetin, myricetin and fisetin. In contrast, cyanidin-3-glucoside, cyanidin-3-sophoroside, cyanidin-3,5-diglucoside, apigenin, luteolin, naringenin and dihydroquercetin were not glucosylated.     

UDP-rhamnose:flavanone-7-O-glucoside-2'-O-rhamnosyltransferase. Purification and characterization of an enzyme catalyzing the production of bitter compounds in citrus

The rhamnosyltransferase catalyzing the production of the bitter flavanone-glucosides, naringin and neohesperidin, was purified to homogeneity. The enzyme catalyzes the transfer of rhamnose from UDP-rhamnose to the C-2 hydroxyl group of glucose attached via C-7-O- of naringenin or hesperetin. To our knowledge this is the first complete purification of a rhamnosyl-transferase. The enzyme from young pummelo leaves was purified greater than 2,700-fold to a specific activity of over 600 pmol/min/mg of protein by sequential column chromatographies on Sephacryl S-200, reactive green 19-agarose, and Mono-Q. The enzyme was selectively eluted from the green dye column with only three other proteins by a pulse of the substrate hesperetin-7-O-glucoside followed by UDP. The rhamnosyltransferase is monomeric (approximately 52 kDa) by gel filtration and electrophoresis. The enzyme rhamnosylates only with UDP-rhamnose. Flavonoid-7-O-glucosides are usable acceptors but 5-O-glucosides or aglycones are not. It is inhibited by 10 microM UDP, its end product, but not by naringin or neohesperidin. Several flavonoid-aglycones at 100 microM inhibited the rhamnosyltransferase; UDP-sugars did not. The Km for UDP-rhamnose was similar with prunin (1.3 microM) and hesperetin-7-O-glucoside (1.1 microM) as substrate. The affinity for the natural acceptor prunin (Km = 2.4 microM) was much higher than for hesperetin-7-O-glucoside (Km = 41.5 microM). The isolation of the gene may enable its use in genetic engineering directed to modifying grapefruit bitterness.     

Flavanone 8-dimethylallyltransferase in Sophora flavescens cell suspension cultures

Dimethylallyl diphosphate: naringenin 8-dimethylallyltransferase (EC 2.5.1) was characterized. The enzyme was enantiospecific for (-)-(2S)-naringenin and utilized 3,3-dimethylallyl diphosphate as sole prenyl donor. It required Mg2+ (optimum concentration, 10 mM), and has an optimum pH of 9-10. The apparent Km values for 3,3-dimethylallyl diphosphate and naringenin were 120 and 36 microM, respectively. The microsomal fraction prenylated several other flavanones at the C-8 position less effectively as compared with naringenin. Interestingly, when 2'-hydroxynaringenin was used as a prenyl acceptor, the 8-lavandulyl (sophoraflavanone G) and the 6-dimethylallyl derivatives were formed, together with the 8-dimethylallyl derivative, leachianone G. These results suggest that the 2'-hydroxy group of naringenin plays an important role for the formation of a lavandulyl group.     

In vitro inhibition of simvastatin metabolism in rat and human liver by naringenin.

We demonstrated that naringenin (NRG), the aglycon form of naringin present in grapefruit juice inhibits in vitro the metabolism of simvastatin (SV), a HMG-CoA reductase inhibitor. SV undergoes an important first pass metabolism and this is thought to be partly responsible for its low bioavailability after oral administration. SV is a prodrug that requires metabolic activation through hydrolysis by esterases. In addition, SV is a substrate for cytochrome P450 enzymes. NRG, a potent inhibitor of cytochrome P450 enzymes, interferes with the isoenzymes of cytochrome P450 involved in the hepatic metabolism of SV. NRG inhibits the metabolism of SV in rat hepatocytes (the intrinsic clearance of SV decreases from 26.2 microl/min/10(6) cells in absence of NRG to 4.15 microl/min/10(6) cells in presence of 50 microM NRG). This inhibition is more pronounced in hepatocytes (Ki value approximately 5 microM) than in liver microsomes (Ki approximately 23 microM and approximately 30 microM in human and rat liver microsomes respectively). Therefore, the hepatocytes seem to be the best approach for in vitro interaction study between SV and NRG ; and this should be taken into account in the in vitro/in vivo extrapolation. If this interaction were confirmed in man, the doses of SV should be reduced when co-administered with grapefruit juice because of increased bioavailability of SV.     

Partial purification, properties, and kinetic studies of UDP-glucose:p-hydroxybenzoate glucosyltransferase from cell cultures of Lithospermum erythrorhizon.

A glucosyltransferase, which catalyzed the transfer of glucose from UDP-glucose (UDPG) to p-hydroxybenzoate (PHB) in cell cultures of Lithospermum erythrorhizon Sieb. et Zucc., Boraginaceae, was purified 219-fold by ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephadex G-150, and phenyl-Sepharose Cl-4B. p-Hydroxybenzoic acid O-beta-D-glucoside (PHB-glc) was identified as a product of the enzymatic reaction. This glucosyltransferase has a molecular weight of 47,500 Da, an isoelectric point at pH 5.0, and a pH optimum of 7.8. The enzyme does not sediment at 100,000g. Enzyme activity did not require metal cofactors. The enzyme was highly specific for p-hydroxybenzoate (Km 0.264 mM) and UDP-glucose (Km 0.268 mM). Initial velocity studies suggest that the enzyme reaction mechanism is a sequential rather than a ping-pong mechanism. Product inhibition patterns are consistent with an ordered sequential bi-bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP the final product released. The data indicate the formation of a dead-end complex between PHB-glc and the enzyme. Uncompetitive inhibition by the substrate PHB can be put down to the formation of an abortive complex between E-UDP and PHB.     

Delay expression of limonoid UDP-glucosyltransferase makes delayed bitterness in citrus

Genes encoding limonoid UDP-glucosyltransferase from albedo of six Citrus species with different levels of delayed bitterness are isolated and cloned in vector pTZ57R/T. Our results indicate that gene sequence of sweet lime (with intense juice delayed bitterness) have complete identity with Satsuma mandarin (without distinctive juice delayed bitterness). Also gene sequence of Marsh seedless grapefruit, local orange and Thompson navel orange (with mild juice delayed bitterness) have very similarity with Satsuma mandarin. On the other hand, this gene started to express 60, 120, and 210 days after full blooming in albedo of Satsuma mandarin, sweet oranges and sour orange, and both grapefruit and sweet lime, respectively. Expression pattern of limonoid glucosyltransferase gene in leaves was quite different with albedo. Thus, we supposed the delayed bitterness in this species was related to delay in expression of limonoid glucosyltransferase gene in albedo and lower limonoid glucoside accumulation in fruits.     

Naringenin is a novel inhibitor of Dictyostelium cell proliferation and cell migration

Naringenin is a flavanone compound that alters critical cellular processes such as cell multiplication, glucose uptake, and mitochondrial activity. In this study, we used the social amoeba, Dictyostelium discoideum, as a model system for examining the cellular processes and signaling pathways affected by naringenin. We found that naringenin inhibited Dictyostelium cell division in a dose-dependent manner (IC(50) approximately 20 microM). Assays of Dictyostelium chemotaxis and multicellular development revealed that naringenin possesses a previously unrecognized ability to suppress amoeboid cell motility. We also found that naringenin, which is known to inhibit phosphatidylinositol 3-kinase activity, had no apparent effect on phosphatidylinositol 3,4,5-trisphosphate synthesis in live Dictyostelium cells; suggesting that this compound suppresses cell growth and migration via alternative signaling pathways. In another context, the discoveries described here highlight the value of using the Dictyostelium model system for identifying and characterizing the mechanisms by which naringenin, and related compounds, exert their effects on eukaryotic cells.     

Bioavailability of the flavanone naringenin and its glycosides in rats

Naringenin, the predominant flavanone in grapefruit, mainly occurs as glycosides such as naringenin-7- rhamnoglucoside or naringenin-7-glucoside. This study compared kinetics of absorption of naringenin and its glycosides in rats either after a single flavanone-containing meal or after adaptation to a diet for 14 days. Regardless of the diet, circulating metabolites were glucurono- and sulfoconjugated derivatives of naringenin. The kinetics of absorption of naringenin and naringenin-7-glucoside were similar, whereas naringenin-7-rhamnoglucoside exhibited a delay in its intestinal absorption, resulting in decreased bioavailability. After naringenin-7-glucoside feeding, no glucoside was found in the cecum. However, after feeding naringenin-7-rhamnoglucoside, some naringenin-7-rhamnoglucoside accumulated in cecum before being hydrolyzed by intestinal microflora. Adaptation to flavanone diets did not induce accumulation of plasma naringenin. Moreover, flavanone cecal content markedly decreased after adaptation, and almost no naringenin-7-rhamnoglucoside was recovered after naringenin-7-rhamnoglucoside feeding, suggesting that an adaptation of cecal microflora had occurred. Overall, these data indicate that flavanones are efficiently absorbed after feeding to rats and that their bioavailability is related to their glycosidic moiety.     

Stereoisomerism in plant disease resistance: induction and isolation of the 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone oxidoreductase, an enzyme introducing chirality during synthesis of isoflavonoid phytoalexins in pea (Pisum sativum L)

Treatment of pea seedlings with CuCl2 induced the activity of the enzyme NADPH:7,2'-dihydroxy-4',5'-methylenedioxyisoflavone oxidoreductase (DMIRase) that introduces (+) stereoisomerism in pisatin. DMIRase was purified approximately 7000 fold from CuCl2-treated pea seedlings to apparent homogeneity by a six-step process. The purification sequence included (NH4)2SO4 fractionation, gel filtration on AcA 44, chromatography on DEAE-Bio-Gel,phenyl-Sepharose CL-4B, and Reactive Red 120-agarose, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration and denaturing electrophoresis showed that the enzyme consisted of a single polypeptide chain with an Mr of 37,500. The pH optimum of DMIRase was determined to be 7.8. The enzyme showed apparent Michaelis constants of 20 microM for 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone and 58 microM for NADPH. The reaction product of the enzyme, sophorol, gave a distinct negative Cotton effect in the region 300-360 nm, which indicated 3S configuration of the molecule. Antibodies against the enzyme were raised in rabbits and characterized for specificity.     

Quantification of the production of dihydrokaempferol by flavanone 3-hydroxytransferase using capillary electrophoresis

A sensitive method using capillary electrophoresis for the separation, detection, and quantification of dihydrokaempferol (1) is reported. Well-resolved, sharp symmetrical peaks were obtained in grapefruit leaf extracts for 1, naringenin (2), and the internal standard, naringin (3). Long columns were required to resolve 1 from 2 in crude enzyme reactions and this resulted in run times of 60 min. The limit of detection for 1 was found to be 1.44 ng/microL (4.2 pg). The method showed excellent linearity and reproducibility. The method was used to determine the activity of flavanone 3-hydroxytransferase (F3H) in leaf tissue of grapefruit by quantification of the production of dihydrokaempferol in controlled time course reactions. The sensitivity of the method makes it adaptable to assaying F3H activity in individual young seedlings and/or in small tissue samples and requires only 100 mg of tissue.