Małgorzata Kalinowska

The Formation of Sugar Chains in Triterpenoid Saponins and Glycoalkaloids

Triterpenoid saponins and structurally related steroidal glycoalkaloids are a large and diverse family of plant glycosides. The importance of these compounds for chemical protection of plants against microbial pathogens and/or herbivores is now well-documented. Moreover, these compounds have a variety of commercial applications, e.g. as drugs or raw materials for pharmaceutical industry. Until recently there were only sparse data on the biosynthesis of saponins and glycoalkaloids, especially at the enzyme level. Substantial progress has recently been made, however, in our understanding of biosynthetic routes leading to the formation of the diverse array of aglycone skeletons found in these compounds as well as mechanisms of synthesis of their sugar moieties. This review highlights some of the advances made over past two decades in our understanding of the formation and modification of sugar moieties in triterpenoid saponins and glycoalkaloids.

Purification and some properties of steryl β-d-glucoside hydrolase from Sinapis alba seedlings

Abstract Homogenates of 7-day-old S. alba seedlings hydrolysed cholesteryl[4-H14C] β- d -glucoside or sitosteryl β- d -glucoside-[6-3H]. Activity was located predominantly in the cell membrane structures sedimenting at 1000–15 000 g and was solubilized by acetone treatment. Partially purified enzyme preparation, with an about 1500 times higher specific activity with respect to the crude homogenate, was obtained by repeated acetone precipitation and subsequent chromatography on DEAE-Sephadex and Sephadex G-100. During this procedure a considerable separation from other enzymes with β-glucosidase activity was achieved. The enzyme had MW 65 000 daltons, pH optimum at 5.2–5.6. Two observations suggested that the enzyme was a specific steryl β- d -glucoside hydrolase. Firstly, there was no substrate competition between steryl glucosides and several other β- d -glucosides. Secondly, enzyme activity wasstrongly inhibited by low concentrations of various 3β-OH sterols with a planar ring system and an intact side chain.

Subcellular localization of udpg: Nuatigenin glucosyltransferase in oat leaves

Abstract 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.

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

Abstract 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.

Enzymatic synthesis of nuatigenin 3β-D-glucoside in oat (Avena sativa) leaves

Abstract Crude homogenates or acetone powder preparations from oat leaves efficiently catalyse the glucosylation of a steroidal sapogenin, nuatigenin [22,25-epoxy-(20 S )(22 S )(25 S )-furost-5-en-3β,26-diol], using UDP-glucose as the sugar donor. The reaction product was identified as nuatigenin 3β- D -monoglucoside. In contrast to the glucosylation of phytosterols, which is also catalysed by enzyme preparations from oat leaves, the formation of nuatigenin glucoside is not stimulated by Triton X-100. This result suggests that glucosyltransferases with different specifirity patterns are involved in sterol and nuatigenin glucosylation in oat leaves. Enzymatic acylation of nuatigenin glucoside to its monoacyl derivative with the use of an endogenous acyl source was also observed with a crude homogenate or a crude membranous fraction as the enzyme preparation.

The occurrence of sterol-ester hydrolase activity in roots of white mustard seedlings

Abstract Roots of white mustard ( Sinapis alba ) seedlings contain sterol-ester hydrolase activity. Enzyme assays with [4- 14 C]cholesteryl palmitate as the substrate show that the hydrolytic activity is located in cell membrane structures but can be easily solubilized with 0.1 % Triton X-100. During gel filtration on Sepharose 6B in the presence of Triton X-100 and sodium chloride the hydrolytic activity is eluted as a single peak. This peak also contains hydrolytic activities toward some other esters such as p -nitrophenyl palmitate, tripalmitoylglycerol or n -hexadecanyl palmitate. However, the rate of hydrolysis of [4- 14 C]cholesteryl palmitate is not significantly affected by a large excess of these esters in the incubation mixtures, suggesting that different enzyme proteins are involved in the hydrolysis of steryl ester and the other esters tested. Sterol-ester hydrolase from white mustard seedlings exhibits a marked specificity with respect to the length of the acyl chain bound to sterol. For a series of steryl esters containing saturated fatty acids (from C 2 to C 22 ) the rate of hydrolysis is the highest for esters of C 14 -C 18 acids. Steryl acetate (C 2 ), butyrate (C 4 ) and behenate (C 22 ) are very poor substrates.

The 3-O-glucosylation of steroidal sapogenins and alkaloids in eggplant (Solanum melongena); Evidence for two separate glucosyltransferases

Abstract UDP-glucose:diosgenin and UDP-glucose:solasodine glucosyltransferase are present in leaves, stems, roots and ripening seeds of garden eggplant (Solanum melongena). These two enzymes share several common properties: i) they occur mainly in the soluble proteins fraction; ii) they co-purify during purification procedure involving ammonium sulphate precipitation, ion-exchange chromatography on Q-Sepharose and gel filtration on Sephadex G-100; iii) they exhibit native Mr values of ca 55 000; iv) they absolutely require reduced -SH groups; and v) they are strongly inhibited by some UDP-glucose analogues such as UDP, UDP-2′,3′-dialdehyde and UDP-mannose. However, the above mentioned enzyme activities can be clearly distinguished using some other effectors. Low concentrations of several non-ionic detergents including Tween 80, Triton X-100 and Tyloxapol almost completely abolish glucosylation of diosgenin but have a slight stimulatory effect on glucosylation of solasodine. Synthetic diosgenin 3-O-β- d -glucopyranoside strongly inhibits glucosylation of diosgenin and some related spirostanols but not that of solasodine. Cholesterol is a potent competitive inhibitor of diosgenin glucosylation (Ki = 1.15 μM) but it has little effect on the glucosylation rate of solasodine. The above data strongly suggest that 3-O-glucosylation of steroidal sapogenins and alkaloids in eggplant leaves is catalyzed by two similar though separate UDP-glucose-dependent glucosyltransferases.

Sterol conjugate interconversions during germination of white mustard (Sinapis alba)

Abstract Changes in the content of free sterols (FS), steryl esters (SE), steryl glucosides (SG) and acylated steryl glucosides (ASG) in germinating seeds of white mustard ( Sinapis alba ) were studied together with parrallel changes in specific activities of some enzymes involved in sterol conjugate transformation. It has been found that a distinct increase in the net SE content and a similar, but less pronounced, increase in SG content at the beginning of germination can be correlated with a distinctly earlier appearance of SE and SG synthesizing enzymes, i.e. triacylglycerol: sterol acyltransferase and UDPG: sterol glucosyltransferase in comparison with hydrolytic activities, i.e. SE hydrolase and SG hydrolase. Our results suggest that metabolism of SG and ASG takes place mainly in the cotyledons while SE metabolism takes place mainly in the roots.

Phospholipids modulate the substrate specificity of soluble UDP-glucose:steroid glucosyltransferase from eggplant leaves

UDP-glucose-dependent glucosylation of solasodine and diosgenin by a soluble, partially purified enzyme fraction from eggplant leaves is affected in a markedly different way by some phospholipids. While glucosylation of diosgenin and some closely related spirostanols, e.g. tigogenin or yamogenin, is strongly inhibited by relatively low concentrations of several phospholipids, the glucosylation of solasodine is unaffected or even slightly stimulated. These effects depend both on the structure of the polar head group and the nature of the acyl chains present in the phospholipid. The most potent inhibitors of diosgenin glucosylation are choline-containing lipids: phosphatidylcholine (PC) and sphingomyelin (SM) but the removal of phosphocholine moiety from these phospholipids by treatment with phospholipase C results in an almost complete recovery of the diosgenin glucoside formation by the enzyme. Significant inhibition of diosgenin glucoside synthesis and stimulation of solasodine glucosylation was found only with PC molecular species containing fatty acids with chain length of 12-18 carbon atoms. PC with shorter or longer acyl chains had little effect on glucosylation of either diosgenin or solasodine. Our results indicate that interaction between the investigated glucosyltransferase and lipids are quite specific and suggest that modulation of the enzyme activity by the nature of the lipid environment may be of importance for regulation of in vivo synthesis of steroidal saponins and glycoalkaloids in eggplant.

Reversible enzymatic transesterification between wax esters and sterols in Sinapis alba roots

Abstract Young roots of mustard ( Sinapis alba ) contain a membrane-bound acyltransferase which catalyses esterification of cholesterol by direct transfer of acyl groups from fatty acid esters of long-chain primary alcohols (wax esters) or esterification of n -hexadecanol with the use of steryl esters as fatty acid sources. Acyl transfer from cholesteryl esters to free cholesterol or from n -hexadecanyl esters to n -hexadecanol can be also observed with this enzyme. The acyltransferase is located in the membranous structures sedimenting at 16 000 g but can be solubilized by acetone treatment and extraction with Tris-maleate, pH 7.3. With the solubilized enzyme the esterification of cholesterol, with n -hexadecanyl palmitate as the acyl source, showed two pH optimaat pH 6.4 and 8.4. In this reaction the best acyl donor among n -hexadecanyl esters varying in the fatty acid moiety was n -hexadecanyl palmitate. Our results suggest that the lsquo;low energy’ exchange of acyl groups may be of importance for steryl ester or/and wax ester synthesis in higher plants.