Isolation and partial characterization of three glucosyl transferases involved in the biosynthesis of flavonol triglucosides in Pisum sativum L

Jourdan, Pablo; Mansell, Richard L.

doi:10.1016/0003-9861(82)90569-0

Cited by 33 publications

(14 citation statements)

References 15 publications

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“…2). Numerous studies have reported monomeric UDP-glycosyl transferases with Mrs ranging from 30 to 68,000 (3,7,22), although multiple subunits and isozymes have also been reported (10,12). Our data indicated the presence of only one form of zucchini GS (Figs.…”

Section: Effects Of Various Metabolitessupporting

confidence: 51%

Purification and Characterization of Galactinol Synthase from Mature Zucchini Squash Leaves

Smith¹,

Kuo²,

Crawford³

1991

Plant Physiol.

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Galactinol synthase catalyzes the first committed step in the biosynthesis of raffinose sugars. Previous attempts to purify the enzyme have proven difficult and have resulted in low quantities of unpurified enzyme. Galactinol synthase was purified 752-fold from mature zucchini (Cucurbita pepo L. cv Burpee Hybrid) leaves using sequential liquid chromatography on DE 52, Octyl-Sepharose CL-4B, and Sephacryl S-200. This isolation scheme resulted in an 18.6% recovery of the initial activity. The purified enzyme had a specific activity of 23.3 micromoles per minute per milligram protein, a pH optimum of 7.5, and the activity was enhanced by dithiothreitol and MnCI2. The enzyme was only half as active with MgCI2 as with MnCI2. Na+, K+, and Ca2+ cations had little effect on the enzyme activity, while Co2+, Zn2+, Cu2 , and Fe3+ cations were strongly inhibitory at 10 millimolar concentrations. Purified galactinol synthase bound specifically to the substrates myoinositol and UDP-galactose (Km = 6.5 and 1.8 millimolar, respectively), while exhibiting little affinity for an alternative glycosyl donor (UDP-glucose) or inositol epimers (epi-and scyllo-). Ten millimolar concentrations of UMP, UDP, UTP, AMP, ADP, ATP, NAD+, NADH, NADP+, UDP-xylose, and UDP-mannose, or 20 millimolar sucrose, talose, galactose, glucose, xylose, and melibiose exhibited various degrees of inhibitory effects. Twenty millimolar stachyose, raffinose, fructose, and mannose, and 10 millimolar UDP-glucuronic acid and UDP-galacturonic acid had little or no effect on the enzyme activity. The purified galactinal synthase is a monomer of Mr 42,000 with an isoelectric point of 4.1.The raffinose family ofoligosaccharides is the second largest group of sugars present in plant tissues (5). In many species, particularly the Cucurbita, these oligosaccharides (raffinose, stachyose, and verbascose) serve as transport sugars (19). Raffinose saccharide biosynthesis is initiated by the transfer of a galactosyl unit from galactinol (O-a-D-galactopyranosyl myo-inositol) to Suc to produce raffinose; this reaction is further iterated to yield stachyose and verbascose (15). Galactinol is synthesized from UDP-Gal and myo-inositol by UDP-D-a-galactose:inositol galactosyltransferase or GS2 (15) as shown below:UDP-Gal + myo-inositol *-* galactinol + UDP 'Supported in part by a grant from the American Soybean Association to T. M. K.

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Section: Effects Of Various Metabolitessupporting

confidence: 51%

Purification and Characterization of Galactinol Synthase from Mature Zucchini Squash Leaves

Smith¹,

Kuo²,

Crawford³

1991

Plant Physiol.

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“…These three compounds are all intermediates in the Ñavonoid biosynthetic pathway. The observed lack of glycosylation of the Ña-vonoid intermediates supports the hypothesis that glycosylation is a terminal step in Ñavonoid biosynthesis, and this has been reported in many studies (Jourdan and Mansell 1982). Sta †ord (1990) reported that some intermediates could be glycosylated for storage, but if they are ever to re-enter the main pathway prior hydrolysis must occur since the Ñavonoid enzymes only function with the aglycones.…”

Section: Resultssupporting

confidence: 58%

“…In many cases these enzymes are able to glycosylate more than one type of B-ring found in anthocyanidins and some can also glycosylate Ñavonols and some other Ña-vonoids, but generally not dihydroÑavonols (Sta †ord 1990). By contrast, novel Ñavonol glycosyltransferase enzymes have been reported from T ulipa anthers (Kleinehollenhorst et al 1982), Pisum Ñowers (Jourdan and Mansell 1982), Chrysosplenium shoots (Bajaj et al 1983) and Anethum cell cultures (Mo hle et al 1985) which exhibited pronounced speciÐcity with regard to the substrate, the position and the sugar transferred (Sta †ord 1990). …”

Section: Introductionmentioning

confidence: 89%

Aglycone and glycoside specificity of apple skin flavonoid glycosyltransferase

Lister

Lancaster

Sutton

et al. 1997

J. Sci. Food Agric.

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O‐Glycosyltransferase(s) extracted from apple (Malus pumila Mill) fruit skin showed activity towards a range of flavonols and anthocyanins. However, no glycosylating activity was shown towards a dihyroflavonol (dihydroquercetin), a flavanone (eriodictyol) or a flavone (luteolin). The enzyme preparation glycosylated those flavonoids normally present in apple skins (quercetin and cyanidin) and in addition several other related compounds (delphinidin, fisetin, isorhamnetin, kaempferol, myricetin and pelargonidin). This enzyme(s) specifically transferred the glycosyl moiety from sugar nucleotide donors to the 3‐position of the flavonoid nucleus. Only flavonoid 3‐glycosides occur naturally in apple skin. Activity with different sugar donors was in the order galactose>glucose>xylose, which reflected the ratios of cyanidin and quercetin glycosides found in apple fruit skin. There were slight differences in the relative UFGT activity with quercetin and the three different sugar donors between ‘Granny Smith’ and ‘Splendour’, and this was reflected by similar differences in the ratios of endogenous quercetin glycosides. ©1997 SCI

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“…In some of its properties, (e.g. Mr and site of glucosylation) the glucosyltransferase from red cabbage resembles those reported from pea (13), Petunia (12,15), Tulipa (16), and parsley cell suspension cultures (24). However, its properties seem to be distinctly different from a cyanidin glucosyltransferase that was reported from illuminated red cabbage seedlings (21).…”

Section: Effect Of Illumination On Enzyme Activitymentioning

confidence: 53%

“…This enzyme preparation was not specific for cyanidin alone, but glucosylated a variety of flavonoid compounds containing 3-hydroxyl groups. The enzyme showed similar properties with glucosyltransferases from other plants such as Petroselinum (24), Chrysosplenium (1), Pisum (13), and Cicer (1 7), that exhibited also a broad substrate specificity.…”

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confidence: 64%

Isolation and Characterization of a UDPGlucose: Flavonol O³-Glucosyltransferase from Illuminated Red Cabbage (Brassica oleracea cv Red Danish) Seedlings

Sun¹,

Hrazdina²

1991

Plant Physiol.

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A UDPGlc:flavonol 03-glucosyltransferase (EC 2.4.1.91) that catalyzes the formation of quercetin and kaempferol 03-glucosides has been purified about 1450-fold from illuminated red cabbage (Brassica oleracea cv Red Danish) seedlings with a 3.3% yield. Purification of the enzyme was achieved by (NH4)2SO4-precipitation, gel-filtration, ion-exchange chromatography on DEAE-Bio-Gel and Q-Sepharose, chromatofocusing, and electrophoresis in nondenaturing polyacrylamide (10%) gels. The enzyme preparation had a pH opfimum between 5.8 and 6.2, isoelectric point in the pH range 4.25 to 4.55, a M, of 59,000, and it was composed of two similar subunits of M, 29,500. The glucosyltransferase reached half substrate saturation at 180 micromolar (UDPGlc) and 7 micromolar (quercetin) concentrations. Kaempferol, which was glucosylated at a relative rate of 87%, had a lesser affinity for the enzyme (Km -12 micromolar). Flavanones, flavanols, flavones, dihydroflavonols, and anthocyanidins were not readily utilized as substrates, suggesting that the enzyme is specific for flavonol glucoside biosynthesis.Glycosylation of flavonoid compounds is thought to occur at or near the end of the biosynthetic pathway (3). The reaction renders a water-soluble product that can be transported to the vacuole, where in general flavonoid glycosides accumulate (9). The most commonly found sugar residue of flavonoid compounds is glucose, located in the 3-position of the molecule, that is transferred from UDPGlc2 to the flavonoid acceptor by the enzyme UDPGlc:flavonoid 03-glucosyltransferase (24).Illuminated red cabbage seedlings synthesize eight anthocyanins (6) that are the major flavonoid constituents of this plant. These are the 3-sophoroside-5-glucoside of cyanidin, and its acylated derivatives with malonic, p-coumaric, ferulic, and sinapic acids (6). While anthocyanins are the most investigated flavonoid components in the Brassicaceae, the presence of flavonol glycosides was also reported (4). Newer reports have confirmed quercetin (5) and kaempferol (23) glycosides as the most commonly occurring flavonol derivatives. Although activation ofenzymes in the phenylpropanoid ' Supported in part by Cornell University and Hatch funds. 2 Abbreviations: UDPGlc, uridine diphosphate glucose; 2-ME, 2-mercaptoethanol; PBE, polybuffer exchanger; PPO, diphenyloxazole; KPi, potassium phosphate. and specific anthocyanin pathway and end-product formation in Brassica was previously investigated in our laboratory, not much is known about the simultaneous synthesis and accumulation of flavonol glycosides.In the metabolism of these compounds, the first glycosylating step is the introduction of glucose into the 3-position of the molecule. Subsequent glucosylations of the introduced sugar and of the hydroxyl group in the 5-position give rise to the 3-sophoroside-5-glucoside of cyanidin and possibly the 3-sophorosides of quercetin and kaempferol.An enzyme that is responsible for glucosylating cyanidin in red cabbage seedlings was reported earlier and purified 50-fol...

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Isolation and partial characterization of three glucosyl transferases involved in the biosynthesis of flavonol triglucosides in Pisum sativum L

Cited by 33 publications

References 15 publications

Purification and Characterization of Galactinol Synthase from Mature Zucchini Squash Leaves

Purification and Characterization of Galactinol Synthase from Mature Zucchini Squash Leaves

Aglycone and glycoside specificity of apple skin flavonoid glycosyltransferase

Isolation and Characterization of a UDPGlucose: Flavonol O³-Glucosyltransferase from Illuminated Red Cabbage (Brassica oleracea cv Red Danish) Seedlings

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Isolation and partial characterization of three glucosyl transferases involved in the biosynthesis of flavonol triglucosides in Pisum sativum L

Cited by 33 publications

References 15 publications

Purification and Characterization of Galactinol Synthase from Mature Zucchini Squash Leaves

Purification and Characterization of Galactinol Synthase from Mature Zucchini Squash Leaves

Aglycone and glycoside specificity of apple skin flavonoid glycosyltransferase

Isolation and Characterization of a UDPGlucose: Flavonol O3-Glucosyltransferase from Illuminated Red Cabbage (Brassica oleracea cv Red Danish) Seedlings

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Isolation and Characterization of a UDPGlucose: Flavonol O³-Glucosyltransferase from Illuminated Red Cabbage (Brassica oleracea cv Red Danish) Seedlings