Purification and Properties of Flavonol-Ring B Glucosyltransferase from <i>Chrysosplenium americanum</i>

Bajaj, K. L.; Luca, Vincenzo De; Khouri, Henry E.; Ibrahim, Ragai K.

doi:10.1104/pp.72.3.891

Cited by 37 publications

(15 citation statements)

References 23 publications

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“…The work described here represents, to our knowledge, the first complete kinetic analysis of a flavonol glucosyltransferase known to catalyze the glucosylation of 2' or 5' positions of partially methylated flavonols [5]. The data obtained from initial velocity and product inhibition studies of this enzyme are consistent with an ordered bi-bi mechanism [9-121; where UDPG is the first substrate that binds to the enzyme, followed by the flavonol compound.…”

Section: Discussionsupporting

confidence: 53%

“…Fractions with high enzyme activity were pooled and used as the enzyme source. Previous purification data [5] showed that the substrate specificity of the Sephadex G-100 fraction was not altered by further purification of the enzyme. Therefore, it was considered appropriate to use the Sephadex G-100 fraction as the enzyme source in this kinetic study.…”

Section: Enzyme Sourcementioning

confidence: 94%

“…The procedure used for enzyme extraction was a modification of that previously described [5]. Unless stated otherwise, all procedures were carried out at 2-4 C. Shoot tips were frozen in liquid nitrogen, mixed with polyclar AT (1 : 10, w/w) and ground to a fine powder.…”

Section: Enzyme Sourcementioning

confidence: 99%

“…1) [4]. A novcl flavonol-ring-B 0-glucosyltransferase, which catalyzed the transfer of glucose from UDPG to positions 2' or 5' of partially methylated flavonols, has been purified from this tissue [5]. It was, therefore, considered pertinent to investigate the kinetic mechanism of glucosylation of these two positions.…”

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confidence: 99%

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Kinetic mechanism of a flavonol‐ring‐B O‐glucosyltransferase from Chrysosplenium americanum

Khouri

Ibrahim

1984

European Journal of Biochemistry

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The kinetic mechanism of a flavonol-ring-B 0-glucosyltransferase from Chrysosplenium americanum was investigated. Substrate interaction kinetics for the flavonol and UDPG gave converging lines, which were consistent with a sequential bireactant binding mechanism. They also showed substrate inhibition with respect to the flavonol. Intercept and slope replots were linear and gave a KA of 250 pM and a KB of 10 pM. Product-inhibition studies showed competitive inhibition between UDPG and UDP (Ki, 20 pM) and non-competitive inhibition between the flavonol substrate and its glucoside (Kip 1 mM). Kinetic patterns were consistent with an ordered bi-bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP is the final product released. The high Kip value, as compared with that of KB, indicates that the reaction is not inhibited by the glucosylated products formed and conforms with the accumulation of flavonol glucosides in C. americanum.

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Section: Discussionsupporting

confidence: 53%

Section: Enzyme Sourcementioning

confidence: 94%

Section: Enzyme Sourcementioning

confidence: 99%

mentioning

confidence: 99%

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Kinetic mechanism of a flavonol‐ring‐B O‐glucosyltransferase from Chrysosplenium americanum

Khouri

Ibrahim

1984

European Journal of Biochemistry

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“…The Mr of the native GTase on gel filtration columns is like that of many other plant glucosyltransferases, approximately 50,000 (29). Whereas some glucosyltransferases are monomeric (1,25), others consist of a dimer of peptide subunits (13,15,16). Thus, peptides Nos.…”

Section: Inductionmentioning

confidence: 99%

Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase in Oat Roots

Yalpani¹,

Balke²,

Schulz³

1992

Plant Physiol.

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A UDP-glucose:salicylic acid 3-0-glucosyltransferase (EC 2.4.1.35) (GTase) from oat (Avena sativa L. cv Dal) root extracts was assayed in vitro using [14C]salicylic acid (SA) and an ion exchange column to separate SA from #-glucosylsalicylic acid. The GTase, present at a very low constitutive level, was inducible to 23 times the constitutive level. When excised roots were exposed to SA at pH 6.5, the specific activity of the enzyme increased within 1.5 h, peaked after 8 to 10 h, and then declined. The increase in specific activity depended on the concentration of SA in the induction medium. Among 16 phenolics and phenolic derivatives tested, GTase induction showed high specificity toward SA and acetylsalicylic acid. Specific activity of the enzyme was induced to higher levels in roots from 7-d-old seedlings than roots from younger plants. GTase the induction of the activity of a GTase (2). The GTase catalyzes the formation of GSA, a compound less inhibitory to Cl-absorption than SA (2). The GTase has been partially purified from oat roots, and some of its properties have been described (2, 29). The protein is soluble, has a native Mr of approximately 50,000, has an isoelectric point of approximately 5, and shows high specificity toward UDP-glucose and SA (29). In this paper, we report our initial studies concerning the induction of GTase activity in oat (Avena sativa L.) roots. MATERIALS AND METHODS Plant Material and ChemicalsOat (Avena sativa L. cv Dal) seeds were germinated on cheesecloth stretched over a 4-L beaker containing 3 L of continuously aerated 10 mm CaSO4 in the dark at room temperature. [7-'4C]SA (1.96 GBq mmolV') and [35S]methionine (>40.7 TBq mmol-') were purchased from New England Nuclear and Amersham, respectively. Induction of GTaseRecently, SA4 was identified as a regulator in the flowering of thermogenic Arum lilies (24) and also as a signal molecule in the activation of resistance responses of plants to pathogens (20, 21). These findings stress the need to understand the processes that regulate SA accumulation. Reports of the formation of SA-glucosides in a number of plants (3,7,17) suggest that SA conjugation could play an important role in determining the size of the free SA pool.Ion uptake by cereal roots is inhibited by exogenous applications of SA (9, 10, 12). Oat roots can recover from this inhibition. It 'Abbreviations: SA, salicylic acid; 2-D, two-dimensional; GSA, ,Bglucosylsalicylic acid; GTase, UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35); mU, milliunit. 1114Roots from 5-d-old seedlings were excised, rinsed with distilled H20, and incubated in 30 mL g-1 of roots of induction buffer (0.5 mm KCl, 0.25 mm CaSO4, 0.5 mm SA, 25 mM Mes/Tris [pH 6.5]) at 250C for the specific times indicated in 'Results.' SA was added in DMSO to a final DMSO concentration of 0.05% (v/v). SA uptake from the induction solution was measured by a spectrophotometric procedure adopted from Brodie et al. (5). Induction solution (1 mL) was mixed with 1.2 mL of 25 mm Fe2(SO4)3...

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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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Purification and Properties of Flavonol-Ring B Glucosyltransferase from Chrysosplenium americanum

Cited by 37 publications

References 23 publications

Kinetic mechanism of a flavonol‐ring‐B O‐glucosyltransferase from Chrysosplenium americanum

Kinetic mechanism of a flavonol‐ring‐B O‐glucosyltransferase from Chrysosplenium americanum

Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase in Oat Roots

Aglycone and glycoside specificity of apple skin flavonoid glycosyltransferase

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