Henry E. Khouri

Enzymology and compartmentation of polymethylated flavonol glucosides in Chrysosplenium americanum

Abstract The enzymatic synthesis of polymethylated flavonol glucosides in Chrysosplenium americanum is catalysed by a number of distinct, position-specific O -methyltransferases and O -glucosyltransferases. These enzymes seem to exist in the form of an aggregated, membrane-associated, multienzyme system. This tissue represents an ideal material for the study of these enzymatic steps, their regulation and compartmentation, as well as the intracellular localization of the final metabolites.

Enzymatic prenylation of isoflavones in white lupin

Abstract Microsomal preparations of white lupin ( Lupinus albus ) radicles and cell suspension cultures catalyse the prenylation of positions 6, 8 and 3′ of the isoflavones genistein and 2′-hydroxygenistein. Both the substrates and the enzyme reaction products are natural constituents of lupin tissues. Enzymatic prenylation of isoflavones required dimethylallyl pyrophosphate (DMAPP) and 12 mM Mn 2+ with optimum activity at pH 7.5 in Tris-HCl buffer. The apparent K m values for DMAPP and the prenyl acceptors were 4 and 5 μM, respectively. Isopentenyl pyrophosphate was a competitive inhibitor of the prenylation reaction, with an apparent K i of 5 μM. The bulk of enzymatic activity was associated with the membrane fraction and could only be solubilized in the presence of a detergent. The differences observed in prenyltransferase activity ratios, in relation to the source of the enzyme and the type of detergent used, suggest that prenylation at positions 6, 8 and 3′ of isoflavones is catalysed by a number of distinct enzymes.

Effects of nutritional and hormonal factors on growth and production of anthraquinone glucosides in cell suspension cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were found to produce anthraquinone glucosides. The effects of nutritional and hormonal factors on growth and anthraquinone production were investigated in order to study the enzymecatalyzed glucosylation of these metabolites.

Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum: III. Purification and kinetic analysis of S-adenosyl-l-methionine:3-methylquercetin 7-O-methyltransferase☆

An O-methyltransferase (OMT) which catalyzes the methylation of 3-methylquercetin to 3,7-dimethylquercetin, the second step of methyl transfers toward the biosynthesis of polymethylated flavonol glucosides, has been isolated from Chrysosplenium americanum shoot tips. The 7-OMT was purified by ammonium sulfate precipitation, gel filtration, chromatofocusing and ion-exchange chromatography using a fast protein liquid chromatography system. Compared with previously reported methods [1985) Arch. Biochem. Biophys. 238, 596-605), this protocol resulted in a highly purified enzyme preparation, free from other OMT activities, which allowed the study of its kinetic mechanism. Substrate interaction and product inhibition patterns obtained were consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind to the enzyme and S-adenosyl-L-homocysteine is the last product released. However, the results obtained did not exclude the formation of one or more dead-end complex. The similarity in kinetic characteristics of this enzyme to those of the other Chrysosplenium OMTs suggests that methyltransferases of this tissue may have evolved from a common precursor.

Partial purification, characterization, and kinetic analysis of isoflavone 5-O-methyltransferase from yellow lupin roots

An isoflavone 5-O-methyltransferase was partially purified from the roots of yellow lupin (Lupinus luteus) by fractional precipitation with ammonium sulfate, followed by gel filtration and ion-exchange chromatography using a fast-protein liquid chromatography system. This enzyme, which was purified 810-fold, catalyzed position-specific methylation of the 5-hydroxyl group of a number of substituted isoflavones. The methyltransferase had a pH optimum of 7 in phosphate buffer, an apparent pI of 5.2, a molecular weight of 55,000, no requirement for Mg2+, and was inhibited by various SH-group reagents. Substrate interaction kinetics of the isoflavonoid substrate and S-adenosyl-L-methionine gave converging lines which were consistent with a sequential bireactant binding mechanism. Furthermore, product inhibition studies showed competitive inhibition between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine and noncompetitive inhibition between the isoflavone and either S-adenosyl-L-homocysteine or the 5-O-methylisoflavone. The kinetic patterns obtained were consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind to the enzyme and S-adenosyl-L-homocysteine is the final product released. The physiological role of this enzyme is discussed in relation to the biosynthesis of 5-O-methylisoflavones of this tissue.

Purification and some properties of five anthraquinone specific glucosyltransferases from Cinchona succirubra cell suspension culture

Abstract Five anthraquinone-specific glucosyltransferases were partially purified from Cinchona succirubra cell suspension culture by fractional precipitation with ammonium sulphate, gel filtration and chromatofocusing on a fast protein liquid chromatography system. Five, distinct glucosylating activities were resolved with apparent pI values of 5.3, 4.8, 4.5, 4.3 and 4.1. They accepted emodin, anthrapurpurin, quinizarin, 2,6-dihydroxy anthraquinone and 1,8- dihydroxy anthraquinone as the best substrates, respectively. These enzymes exhibited similar characteristics as to pH optimum (pH 7) in histidine/HCl buffer, M , 50 000, had no cation requirement and were inhibited by various SH-group reagents. The K m value of the respective anthraquinones for either of the five enzymes was 10 μM. The physiological role of these novel enzymes is discussed in relation to the biosynthesis of anthraquinone glucosides in this tissue.

Resolution of five position-specific flavonoid o-methyl-transferases by fast protein liquid chromatofocusing

Abstract The enzymatic synthesis of polymethylated flavonol glucosides in Chrysosplenium americanum is catalysed by a number of distinct, position-specific

Fast protein liquid chromatographic purification and some properties of a partially O-methylated flavonol glucoside 2'-/5'-O-methyltransferase

Abstract A flavonol O -methyltransferase was partially purified from Chrysosplenium americanum by fractional precipitation with ammonium sulphate followed by gel filtration and chromatofocusing using an FPLC system. The enzyme which was purified 420-fold catalysed the transfer of the methyl group of SAM to the 2′- or 5′-positions of partially methylated flavonol glucosides, the two terminal methylation steps in the biosynthesis of Chrysosplenium flavonoids. The enzyme had a pH optimum of 7 in Pi buffer, a pI of M , of 57 000, no Mg 2+ requirement and was inhibited by both N -ethylmaleimide and phenylmercuriacetate. The K m value for the flavonol substrate was 2 μM and that for SAM was 100 μM. The role of this enzyme is discussed in relation to the biosynthesis of polymethylated flavonols in this tissue.

Flavonol 3-O-MethyItransferase in Plant Tissues

Flavonol 3-O-methyltransferase activity has been detected in Calamondin orange peel and root tissue, tobacco cell culture and young shoots of Chrysosplenium americanum. The enzyme was purified from the latter tissue by precipitation with ammonium sulphate, ion-exchange chromatography and finally by chromatofocusing on Poly- buffer exchanger at pi 4.8. The focused enzyme exhibited strict stereospecificity towards quercetin, MW of 65000 daltons and pH optimum of 7.5-8.5. The apparent Km values for quercetin and S-adenosyl-ʟ-methionine were 1.4 and 70 μM, respectively.