Jan Zimowski

Specificity of sterol-glucosylating enzymes from Sinapis alba and Physarum polycephalum

1. Sinapis alba L. seedlings contain glycosyltransferase catalyzing the synthesis of sterol glucosides in the presence of UDPglucose as sugar donor. The major activity occurs in the membranous fraction sedimenting at 300--9000 x g. Successive treatment of the particulate enzyme fraction with acetone and Triton X-100 affords a soluble glucosyltransferase preparation which can be partly purified by gel filtration on Sephadex G-150. Molecular weight of the glucosyltransferase is 1.4 . 10(5). Apparent Km values for UDPglucose and sitosterol are 8.0 . 10(-5) M and 5.0 . 10(-6) M, respectively. 2. Comparison was made of the S. alba glucosyltransferase with a similar sterol-glucosylating enzyme isolated from non-photosynthesizing organism Physarum polycephalum (Myxomycetes). UDPglucose was the most efficient glucose donor in both cases but the enzyme from Ph. polycephalum can also utilize CDPglucose and TDPglucose. Glucose acceptors are, in case of both enzymes, sterols containing a beta-OH group at C-3 and a planar ring system (5 alpha-H or double bond at C-5). The number and position of double bonds in the ring system and in the side chain, as well as the presence of additional alkyl groups in the side chain at C-24 are of secondary importance. 3. The present results indicate that both enzymes can be regarded as specific UDPglucose:sterol glucosyltransferases. Certain differences in their specificity towards donors and acceptors of the glucosyl moiety suggest, however, a different structure of the active sites in both enzymes.

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.

Occurrence of a glucosyltransferase specific for solanidine in potato plants

Abstract It was found that potato tubers, sprouts, leaves and stems show the presence of two different enzymic activities responsible for steroid glucosylation and utilization of UDPG as a donor of the glucose residue. The first is a typical UDPG : sterol glucosyltransferase bound to the membranes (105 000 g pellet) which is strongly stimulated by 0.1% Triton X-100. The second enzyme is a cytosolic glucosyltransferase which is slightly inhibited by the detergent, and which shows the ability to glycosylate solanidine (22 S ,25 S -solanid-5-en-3β-ol) with a high yield. The molecular weight of the latter enzyme was determined by molecular filtration on Sephadex G-150 to be ca 50 000. Moreover, it was found that the cytosolic enzyme preparation also catalyses solanidine galactosylation, although with a much lower yield, using UDP-galactose as the source of the sugar residue.

Specificity and some other properties of cytosolic and membranous udpglc: 3β-hydroxysteroid glucosyltransferases from Solanum tuberosum leaves

Abstract Our earlier studies have suggested that potato plant leaves contain two UDPGlc-dependent glucosyltransferases: the membranous enzyme with high affinity to sitosterol and cytosolic enzyme with high affinity to solanidine. Some properties of both these glucosyltransferases have been compared and their specificity for various 3β-hydroxysteroids representing different types of structure examined in detail. Both enzymes displayed the ability to glucosylate the examined steroid substrates. The membranous enzyme glucosylated them in the following sequence: typical plant sterols > androstane and pregnane derivatives > steroid alkaloids of the spirosolane type and steroid sapogenins > steroid alkaloids of the solanidane type. The cytosolic enzyme glucosylated the steroid substrates in the order: steroid alkaloids of the solanidane type > steroid alkaloids of the spirosolane type > steroid sapogenins > sterols, pregnane and androstane derivatives. It was found that glucosylation of steroid compounds by the membranous enzyme was stimulated by 0.1% Triton X-100, whereas glucosylation catalysed by the cytosolic enzyme was inhibited in the presence of this detergent. Both enzymes displayed a similar optimum pH (6.5–7.0) and did not require divalent ions. The cytosolic enzyme, in contrast to the membranous one, was somewhat stimulated by 2-mercaptoethanol and inhibited in the presence of high ionic strength. The results afforded additional evidence that potato plant leaves contain, apart from the membranous UDPGlc:sterol glucosyltransferase resembling that occurring in many other higher plants, the enzyme preferentially glucosylating solanidine, which may be involved in initiation of the synthesis of the sugar chains in glycoalkaloids of the α-chaconine series.

Partial purification and specificity of triacylglycerol: Sterol acyltransferase from Sinapis alba

Abstract Triacylglycerol: sterol acyltransferase is present in roots of Sinapis alba seedlings. The enzyme is located predominantly in the cell membrane structures sedimenting at 300–16 000 g but can be solubilized by acetone treatment and buffer extraction. During gel filtration on Sephadex G-100 the acyltransferase activity was separated into two peaks corresponding to MW 1.8 × 10 14 and MW ⩾ 10 5 , respectively. A number of natural 3β-hydroxysterols can be esterified by the solubilized acyltransferase. The rate of esterification is much higher for sterols containing a planar ring system. The number and position of double bonds, as well as the structure of the side chain at C- 17 of the sterol molecule, are of secondary importance. Triacylglycerols containing fatty acids C, C 6 -C 22 can be utilized as acyl donors. Among triacylglycerols containing saturated fatty acids, tripalmitoylglycerol (C 16:0 ) is the best acyl donor. For triacylglycerols containing C 18 -fatty acids the following sequence was observed: trioleoylglycerol (C 18:1 ) > trilinoleoylglycerol (C 18:2 ) > trilinolenoylglycerol (C 18:3 ) > tristearoylglycerol (C 18:0 ).

Acyl donors for sterol esterification by cell-free preparations from Sinapis alba roots

Abstract Homogenates or crude 300-16 000 g membrane fractions from Sinapis alba roots catalysed esterification of [4- 14 C]cholesterol with utilization of endogenous acyl sources. With acetone powder preparations cholesterol esterification was distinctly stimulated by a neutral lipid fraction isolated from S. alba roots. Among neutral lipids triacylglycerols were the most active in this process. Experiments with various acyl-labelled acylglycerols as acyl donors and non-labelled sterols as acceptors confirmed that triacylglycerols are directly utilized as the source of fatty acids for sterol esterification. Di- and mono-acylglycerols were much less effective.

Acyl composition and biosynthesis of acylated steryl glucosides in Calendula officinalis

Fatty acids C12-C22 are components of acylated steryl glucosides in Calendula officinalis. Various particulate fractions from 14-day-old seedlings catalyze the esterification of the steryl glucosides with utilization of endogenous acyl donors. The activity seems to be associated mainly with the membranous structures being fragments of Golgi complex, as it has previously been suggested for UDPG: sterol glucosyltransferase. Succesive treatment of the particulate enzyme fraction with Triton X-100 and acetone affords a soluble acyltransferase preparation partly depleted of endogenous lipids. As a source of acyl groups for the synthesis of steryl acylglucosides this preparation utilizes various phospholipids obtained from the same plant in the following sequence: phosphatidylinositol greater than phosphatidylethanolamine greater than phosphatidylcholine. It does not utilize triacylglycerols and monogalactosyldiacylglycerols.

Steroid-specific glucosyltransferases in Asparagus plumosus shoots

Abstract Lipid-depleted enzyme preparations (‘acetone powders’) obtained from young shoots of Asparagus plumosus efficiently catalyse UDPG-dependent glucosylation of many 3β-hydroxy steroids including phytosterols, steroidal sapogenins of the spirostane type and some steroidal alkaloids structurally related to spirostanols. Some differences in subcellular distribution of enzyme activities, as measured with various steroid acceptors, and clearly different effects of Triton X-100 on individual reactions, indicate the presence of at least two distinct steroid-specific glucosyltransferases. In addition to the well-known UDPG: sterol glucosyltransferase which is, as in many other vascular plants, a tightly membrane-bound, Triton-activated enzyme, A. plumosus shoots contain another UDPG-dependent glucosyltransferase which seems to be less specific for steroid acceptor. The latter enzyme is found both in the membranous and soluble fractions and is distinctly inhibited by Triton X-100. It efficiently glucosylates yamogenin (the aglycone of steroidal saponins present in A. plumosus) to yamogenin 3-monoglucoside. This reaction product can be regarded as an intermediate in the biosynthesis of spirostane and furostane saponins present in A. plumosus.

Enzymatic synthesis of steryl 3β-d-monoglucosides in the slime mold Physarum polycephalum

Abstract Microplasmodia of Physarum polycephalum exhibit high UDPG: sterol glucosyltransferase activity. The enzyme was purified about 28-fold by acetone pr

Specificity and properties of UDP-galactose:tomatidine galactosyltransferase from tomato leaves

Abstract The specificity of galactosyltransferase involved in the biosynthesis of tomatidine monogalactoside in tomato leaves was studied using a wide spectrum of 3-OH steroids (i.e. steroidal alkaloids of spirosolane and solanidane type, steroidal sapogenins, typical sterols, androstane, pregnane and cholesterol derivatives as well as triterpenic alcohols) as sugar acceptors. The highest activity was found with tomatidine, but some other structurally-related compounds such as solasodine, nuatigenin, isonuatigenin, hecogenin, tigogenin, diosgenin were also glycosylated, however, at lower rates. UDP-galactose appeared to be the best sugar donor. The enzyme preparation was also able to utilize UDP-glucose as a sugar source for tomatidine glucosylation, however, at distinctly lower rate. Kinetic data showed apparent K m values of 2.48 μ M for UDP-galactose and 0.83 μ M for tomatidine. The investigated galactosyltransferase was stimulated by 2-mercaptoethanol and inhibited in the presence of UDP, UTP, divalent metal ions such as Cu 2+ , Zn 2+ , Hg 2+ , Triton X-100, high ionic strength and N -ethylmaleimide. Divalent metals ions such as Mg 2+ , Ca 2+ , Mn 2+ and chelating agents (EDTA, EGTA) had no significant effect on the enzyme activity.