Purification and Properties of Flavonol - Ring B

A novel glucosyltransferase which catalyzed the transfer of glucose from UDP-glucose to positions 2' and 5' of partially methylated flavonols was isolated from the shoots of Chrysosplenium americanum Schwein ex Hooker. It was purified 225-fold by ammonium sulfate precipitation and successive chromatography on Sephadex G-100, hydroxyapatite, and polybuffer ion exchanger. This glucosyltransferase appeared to be a single polypeptide with an apparent molecular weight of 42,000 daltons, pH optimum of 7.5 to 8.0, and an isoelectric point of 5.1. It had low but similar Km values for the 2' and 5' positions of flavonol substrates and the cosubstrate UDP-glucose and was inhibited by both reaction products, the glucosides formed, and UDP. Glucosyltransferase activity was independent of divalent cations, was not inhibited by EDTA, but showed requirement for SH groups. The differential effect on enzyme activity of metal ions, especially cupric ion, and various SH group reagents seemed to indicate the involvement of two active sites in the glucosylation reaction; the site specific for 2' activity being more susceptible than that of the 5' activity. The substrate specificity expressed by this glucosyltransferase and the requirement of at least two para-oriented B-ring substituents (at 2' and 5') for activity support this view. The transfer of the glucosyl moiety from sugar nucleotides to flavonoid acceptors has been widely studied (12) and is considered to be a terminal step in flavonoid biosynthesis (9, 10). Whereas glucosyltransferases were believed to possess a broad substrate specificity, recent reports tend to indicate their specificity towards the different classes of flavonoids, e.g. flavones, isoflavones, flavonols, and anthocyanidins (18, 22, 23). Position specificity has also been reported for the 7-O-glucosyltransferases of flavones, isoflavones, and flavonols (18, 23, 24), as well as the much investigated 3-O-glucosyltransferases of flavonols and anthocyanidins (21-24). No glucosyltransferase specific for ring B of flavonoids has, so far, been reported except for the detection of a weak glucosylating activity for positions 5, 7, and 3' of the flavonol quercetin by nonpurified extracts of Zea mays (7). Chrysosplenium americanum, a Saxifragaceae, contains two 2'O-D-glucosides of partially methylated 2'-hydroxyquercetin and four 5'-O-D-glucosides of partially methylated 6-hydroxyand 6,2'-dihydroxyquercetin (Fig. 1; Ref. 4). Our current interest in the enzymic synthesis of partially methylated flavonols (4-6, 15) 'Supported by Grant No. A4549 from the Natural Science and Engineering Research Council of Canada. 2Present address: Department of Biochemistry, Punjab Agricultural University, Ludhiana, 141004 India. 'To whom request for reprints should be addressed. prompted us to study the glucosyltransferase(s) involved in their biosynthesis. In view of the position specificity exhibited by a number of O-methyltransferases isolated from this plant (6), it was interesting to find out whether glucosylation of the 2' and 5' positions was catalyzed by one or two distinct enzymes. We wish to report in this paper on the purification and some properties of a novel enzyme which catalyzed the transfer of glucose from UDP-glucose to positions 2' and 5' of partially methylated flavonols in C. americanum. MATERIALS AND METHODS Plant Material. Chrysosplenium americanum Schwein ex Hooker (Saxifragaceae) was collected from Sutton junction, Eastern Townships, Province ofQuebec, and was maintained in the greenhouse under natural light at 18 to 22°C. Chemicals. UDP-[U-'4CJglucose (334 mCi/mmol) was obtained from New England Nuclear; UDP, UDPG, N-ethylmaleimide, pchloromercuribenzoate, and Dowex chelating resin were purchased from Sigma. Sephadex G-25, G-100, DEAE-Sephadex A25, polybuffer ion exchanger PBE-94, polybuffer PB-74, and the reference proteins used for mol wt determination were from Pharmacia Fine Chemicals. Hydroxyapatite (Bio-gel HT) was from Bio-Rad, and DEAE-cellulose (DE-52) was from Whatman Chemical Separation Ltd, England. Flavonoid substrates and reference compounds were from our laboratory collection and from those previously isolated and identified (4). Imidazole was recrystallized from ethylacetate before use. Enzyme Extraction and Purification. Unless stated otherwise, all procedures were carried out at 2 to 4°C. About 15 g of shoot tips were frozen in liquid N2, mixed with Polyclar AT (1:5, w/w), and ground to a fine powder. The mixture was homogenized with 0.2 M Tris-HCl buffer (pH 7.6; 1:4, w/v), containing 14 mm 2mercaptoethanol, 5 mm EDTA, and 10 mm diethylammonium diethyldithiocarbamate (buffer A). The homogenate was filtered through nylon mesh and the filtrate was centrifuged at 15,000g for 15 min. The supernatant was stirred for 20 min with Dowex 1x2 (20%7b, w/v) which had previously been equilibrated with buffer A, then filtered through glass wool. The filtxrate was fracOMe Compound R1 R2 R3 Glucose Me 0 R3 A H OH OH R2 B H OH OMe R2 'Me c OH H OH R3 D Ome H R3 OH 0 E OMe OH OH R3 F OMe OMe OH R3 FIG. 1. Structural formulae of the partially methylated flavonol aglycones (A-F) used as substrates for the glucosyltransferase assay; their 2' (R2) and 5' (R3) glucosides occur naturally in C. americanum. Note that whenever the 2' position is substituted then numbering of the o-dihydroxy grouping becomes 4',5'instead of 3',4'-, since the 3'and 5'-positions are identical. 891 www.plantphysiol.org on July 22, 2017 Published by Downloaded from Copyright © 1983 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 72, 1983