Purification and characterization of a novel glucosyltransferase specific to 27beta-hydroxy steroidal lactones from Withania somnifera and its role in stress responses

Sterol glycosyltransferases catalyze the synthesis of diverse glycosterols in plants. Withania somnifera is a medically important plant, known for a variety of pharmacologically important withanolides and their glycosides. In this study, a novel 27beta-hydroxy glucosyltransferase was purified to near homogeneity from cytosolic fraction of W. somnifera leaves and studied for its biochemical and kinetic properties. The purified enzyme showed activity with UDP-glucose but not with UDP-galactose as sugar donor. It exhibited broad sterol specificity by glucosylating a variety of sterols/withanolides with beta-OH group at C-17, C-21 and C-27 positions. It transferred glucose to the alkanol at C-25 position of the lactone ring, provided an alpha-OH was present at C-17 in the sterol skeleton. A comparable enzyme has not been reported earlier from plants. The enzyme is distinct from the previously purified W. somnifera 3beta-hydroxy specific sterol glucosyltransferase and does not glucosylate the sterols at C-3 position; though it also follows an ordered sequential bisubstrate reaction mechanism, in which UDP-glucose and sterol are the first and second binding substrates. The enzyme activity with withanolides suggests its role in secondary metabolism in W. somnifera. Results on peptide mass fingerprinting showed its resemblance with glycuronosyltransferase like protein. The enzyme activity in the leaves of W. somnifera was enhanced following the application of salicylic acid. In contrast, it decreased rapidly on exposure of the plants to heat shock, suggesting functional role of the enzyme in biotic and abiotic stresses.     

Steryl glucoside biosynthesis in the alga Prototheca zopfii

A particulate enzyme fraction from the Chlorophyta Prototheca zopfii catalysed the transfer of glucose-[U-¹⁴C]from UDP-Glc-[U-¹⁴C] to endogenous sterol acceptors and the esterification of steryl glucosides with fatty acids from an endogenous acyl donor. Glucose was the only sugar present, and it appeared to have the β-configuration. In the acylated derivatives the glucose-acyl linkage appeared in the C-6 position of glucose, as indicated by periodate oxidation. UDP-Glc:sterol glucosyltransferase was solubilized with detergent and purified 34-fold. The solubilized enzyme showed no specificity for the sterol but a high affinity for the sugar nucleotide UDP-Glc. Time-course incorporation into steryl glucoside (SG) and the acylderivative (ASG) indicated that SG was the precursor of ASG and that phosphatidyl ethanolamine stimulated the formation of the latter compound, presumably acting as acyl donor. A high sterol glucosylating activity was found in the Golgirich fraction. All this evidence indicates that steryl glucosides and their acylated derivatives were synthesized by algae. The early assumption that these compounds were not present in algae must be revised.     

Enzymatic deoxyglucosylation of ceramides by microsomes of BHK-21 cells. The effect of deoxyglucose treatment and herpesvirus infection

The microsomal fractions of cultured hamster fibroblasts (BHK-21 cells) catalyze the incorporation of glucose from UDPglucose or of deoxyglucose from UDPdeoxyglucose into a reaction mixture with liposomes consisting of ceramide and phosphatidylcholine. The microsomal fractions also catalyze the transfer of glucose from UDPglucose to endogenous acceptors. The specific activity of ceramide deoxyglucoside or ceramide glucoside formation was significantly higher when microsomal preparations obtained from deoxyglucose-treated or herpesvirus-infected BHK-21 cells were used as the glucosyltransferase source. Deoxyglucose was incorporated from UDPdeoxyglucose into hydroxy- and nonhydroxy-fatty acid-containing ceramides at approximately the same rate. Competitive inhibition of deoxyglucosylation of ceramides by UDPglucose suggests that both reactions were catalyzed by the same enzyme, viz. UDPglucose:ceramide glucosyltransferase. This inhibition of glycosphingolipid synthesis may account, in part, for the inhibitory effect of deoxyglucose on lipid-containing viruses.     

Characterization of human platelet UDPglucose-collagen glucosyltransferase using a new rapid assay.

A rapid and specific assay has been developed for UDPglucose-collagen glucosyltransferase (UDPglucose: 5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) using galactosylhydroxylysine (Gal-Hyl) as acceptor. Studies with intact human platelets and isolated plasma membranes indicated that about 5--10% of the total activity was surface bound and the rest was of cytoplasmic origin. The two forms of the enzyme had similar broad pH optima (6.5--8.0), Km values for UDPglucose (5 muM) and Gal-Hyl (approx. 4 mM) and for optimal manganese concentrations (25 mM). The soluble form of the enzyme was purified 80-fold. The reaction mechanism was determined as being rapid equilibrium random BiBi + dead end complex or ordered BiBi with UDPglucose being the first substrate to bind. Using Gal-Hyl bound in purified alpha 1 chain of chick skin collagen, a Km value three orders of magnitude less (2 muM) was found than for free Gal-Hyl and the manganese requirement decreased to 2 mM. These results suggest that the binding to the enzyme of Gal-Hyl in the collagen molecule is enhanced by the presence of the protein portion so that the enzyme may be capable of recognizing not only the carbohydrate side chains but also the primary structure of collagen.     

Is Pneumocystis a Plant?

Pneumocystis, an AIDS-associated opportunistic pathogen of the lung has some unusual features. This article focuses on work done by my group to understand the organism's distinct sterols. Although Pneumocystis is closely related to fungi, it lacks the major fungal sterol, ergosterol. Several delta(7) 24-alkysterols synthesized by P. carinii are the same as those reported in some basidiomycete rust fungi. The 24-alkylsterols are synthesized by the action of S-adenosyl-L-methionine:C-24 sterol methyl transferase (SAM:SMT). Fungal SAM:SMT enzymes normally transfer only one methyl group to the C-24 position of the sterol side chain and the cells accumulate C28 24-alkylsterols. In contrast, the P. carinii SAM:SMT and those of some plants catalyze one or two methyl transfer reactions producing both C28 and C29 24-alkylsterols. However, unlike most fungi, plants, and the kinetoplastid flagellates Leishmania and Trypanosoma cruzi, P. carinii does not appear to form double bonds at C-5 of the sterol nucleus and C-22 of the sterol side chain. Furthermore, the P. carinii SAM:SMT substrate preference for C30 lanosterol differs from that of homologous enzymes in any other organisms studied. C31 24-Methylenelanosterol and C32 pneumocysterol, products of SAM:SMT activity on lanosterol, can accumulate in high amounts in some, but not all, human-derived Pneumocystis jiroveci populations.     

Cholesterol oxidase susceptibility of cholesterol and 5-androsten-3 beta-ol in pure sterol monolayers and in mixed monolayers containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

This study has examined the importance of the isocaproic side chain at C-17 of cholesterol to sterol/phospholipid interactions in monolayer membranes and to the cholesterol oxidase-susceptibility of cholesterol in pure and mixed monolayers at the air/water interface. The interactions between cholesterol or 5-androsten-3 beta-ol (which lacks the C-17 side chain) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in monolayers indicated that 5-androsten-3 beta-ol was not very efficient in causing condensation of the monolayer packing of POPC. Whereas cholesterol condensed the packing of POPC at all molar fractions examined (i.e., 0.25, 0.50 and 0.75 with regard to POPC), 5-androsten-3 beta-ol caused a slight condensing effect on POPC packing only in the equimolar mixture. The mean molecular area requirement of 5-androsten-3 beta-ol (in pure sterol monolayers at different lateral surface pressures) was 2.2-6.7% less than that observed for cholesterol. The pure 5-androsten-3 beta-ol monolayer also collapsed at lower lateral surface pressures compared with the pure cholesterol monolayer (34 mN/m and 45 mN/m, respectively). The cholesterol oxidase (Streptomyces sp.) catalyzed oxidation of cholesterol or 5-androsten-3 beta-ol in pure monolayers in the air/water interface (10 mN/m) proceeded with very similar rates, indicating that the enzyme did not recognize that the C-17 side chain of 5-androsten-3 beta-ol was missing. The oxidation of cholesterol or 5-androsten-3 beta-ol in mixed POPC-containing monolayers (equimolar mixture) also revealed similar reaction rates, although the reaction was slower in the mixed monolayer compared with the pure sterol monolayer. When the oxidation of cholesterol and 5-androsten-3 beta-ol was examined by monitoring the production of H2O2 (the sterol was solubilized in 2-propanol and the assay conducted in phosphate buffer), the maximal reaction rate observed with 5-androsten-3 beta-ol was only about 41% of that measured with cholesterol. From the cholesterol oxidase point-of-view, it can be concluded that the enzyme did not recognize the C-17 side chain of cholesterol (or lack of it in 5-androsten-3 beta-ol), when the sterol was properly oriented as a monolayer at the air/water interface. However, when the substrate was presented to the enzyme in a less controlled orientation (organic solvent in water), 5-androsten-3 beta-ol may have oriented itself unfavorably compared with the orientation of cholesterol, thereby leading to slower oxidation rates.     

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

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 × 10¹⁴ and MW ? 10⁵, 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, C₆-C₂₂ can be utilized as acyl donors. Among triacylglycerols containing saturated fatty acids, tripalmitoylglycerol (C_16:0) is the best acyl donor. For triacylglycerols containing C₁₈-fatty acids the following sequence was observed: trioleoylglycerol (C_18:1) > trilinoleoylglycerol (C_18:2) > trilinolenoylglycerol (C_18:3) > tristearoylglycerol (C_18:0).     

FadD19 of Rhodococcus rhodochrous DSM43269, a steroid-coenzyme A ligase essential for degradation of C-24 branched sterol side chains

The actinobacterial cholesterol catabolic gene cluster contains a subset of genes that encode β-oxidation enzymes with a putative role in sterol side chain degradation. We investigated the physiological roles of several genes, i.e., fadD17, fadD19, fadE26, fadE27, and ro04690DSM43269, by gene inactivation studies in mutant strain RG32 of Rhodococcus rhodochrous DSM43269. Mutant strain RG32 is devoid of 3-ketosteroid 9α-hydroxylase (KSH) activity and was constructed following the identification, cloning, and sequential inactivation of five kshA gene homologs in strain DSM43269. We show that mutant strain RG32 is fully blocked in steroid ring degradation but capable of selective sterol side chain degradation. Except for RG32ΔfadD19, none of the mutants constructed in RG32 revealed an aberrant phenotype on sterol side chain degradation compared to parent strain RG32. Deletion of fadD19 in strain RG32 completely blocked side chain degradation of C-24 branched sterols but interestingly not that of cholesterol. The additional inactivation of fadD17 in mutant RG32ΔfadD19 also did not affect cholesterol side chain degradation. Heterologously expressed FadD19DSM43269 nevertheless was active toward steroid-C26-oic acid substrates. Our data identified FadD19 as a steroid-coenzyme A (CoA) ligase with an essential in vivo role in the degradation of the side chains of C-24 branched-chain sterols. This paper reports the identification and characterization of a CoA ligase with an in vivo role in sterol side chain degradation. The high similarity (67%) between the FadD19(DSM43269) and FadD19H37Rv enzymes further suggests that FadD19H37Rv has an in vivo role in sterol metabolism of Mycobacterium tuberculosis H37Rv.     

The functional importance of structural features of ergosterol in yeast.

As an approach to the study of the relationship between the structure of sterols and their capacity to function in the lipid leaflet of membranes, various sterols were examined for their ability to support the growth of anaerobic Saccharomyces cerevisiae. A marked dependence on precise structural features was observed in growth-response and morphology. Of the chemical groups which distinguish ergosterol, the main sterol of S. cerevisiae, the hydroxyl group at C-3 was obligatory, and the other groups were found to be of the following relative importance: 24beta-methyl-delta22-grouping greater than 24beta-methyl group greater than delta5,7-diene system = delta5-bond approximately or equal to no double bond. Methyl groups at C-4 and C-14 were inconsistent with activity. Consequently, the data strongly suggest that the normal biosynthetic processes removal of methyl groups from the nucleus and introduction of one in the side chain are of functional significance. A double bond between C-17 and C-20 joining the steroidal side chain to the nucleus had no deleterious effect on the growth process but only if C-22 was trans-oriented to C-13. In the cis-case no growth at all proceeded. This means the natural sterol probably acts functionally in the form of its preferred conformer in which C-22 is to the right ("right-handed") in the usual view. Since the placing of a substituent (OH or CH3) in the molecule at C-20 in such a way that it appears on the front side in the right-handed conformer completely destroyed activity, the sterol apparently presents its front face to protein or phospholipid when complexing occurs.     

A radioassay of enzymes catalyzing the glucosylation and sulfation steps of glucosinolate biosynthesis in Brassica species

A new method for assaying the enzymes uridine diphosphoglucose (UDPglucose):thiohydroximate glucosyltransferase and 3'-phosphoadenosine-5'-phosphosulfate:desulfoglucosinolate sulfotransferase has been designed. The assay system is based on the separation of nonionic [14C]desulfobenzylglucosinolate from anionic [14C]UDPglucose and anionic [14C]benzylglucosinolate, respectively, by differential adsorption to DEAE-ion-exchange disks. The procedure eliminates elaborate chromatographic techniques. The method was used to measure both enzymes in several Brassica spp. In addition, sulfotransferase activity was monitored during partial purification from seedlings of Brassica napus (cv Westar).     

Steroids in Porifera. II. Steroid derivatives from two sponges of the family Halichondriidae. Sokotrasterol sulfate, a marine steroid with a new pattern of side chain alkylation

A trisulfated derivative of 24,25,26,26-tetramethyl-5 alpha-cholest-23E-ene-2 alpha, 3 beta, 6 alpha-triol (sokotrasterol sulfate) has been isolated from the sponge Halichondriidae gen. sp., collected near Sokotra Island (Arabian Sea), and its structure has been elucidated. The side chain of the new steroid involves a "normal" alkylation at C-24 and the unprecedented addition of two extra methyl groups at C-26 and one extra methyl group at C-25. A free sterol fraction contained only 24-isopropyl-5-cholesten-3 beta-ol and 24-isopropyl-5, 22E-cholestadien-3 beta-ol. 24-Isopropyl-5, 22E-cholestadien-3 beta-ol as sole monohydroxy sterol and halistanol sulfate as major polyhydroxylated steroid derivative have been detected in Halichondria sp., a Madagascar sponge.     

76 The Pneumocystis sam:smt, an atypical fungal enzyme protein

Pneumocystis , an opportunistic fungal protist, causes a type of pneumonia in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii and human-derived P. jiroveci contain a large number of sterols with C-24 alkyl groups. S-Adenosyl-L-methionine:sterol C-24 methyl transferase (SAM:SMT) is the enzyme that transfers methyl groups from SAM to the C-24 position of the sterol side chain. An alkyl group at the C-24 sterol side chain position appears to be essential for the organism to proliferate. Thus SAM:SMT, which is absent in mammals, is an attractive target for chemotherapeutic attack against the pathogen. The P. carinii erg6 gene that codes for SAM:SMT has been sequenced, cloned, and the protein expressed in E. coli . Since bacteria do not synthesize sterols, and do not have SAM:SMT, the P. carinii erg6 gene product expressed in E. coli would only transmethylate exogenously provided sterol substrates. The P. carinii recombinant SAM:SMT is unique because lanosterol, a central intermediate in sterol biosynthesis, is its preferred substrate for enzyme activity. Most SAM:SMT from other organisms do not bind lanosterol and prefer other sterol substrates produced from lanosterol. Furthermore, it appears that this unusual P. carinii SAM:SMT can also methylate cholesterol, which is readily scavenged from the lungs of its rat host. The recombinant enzyme protein is being purified by affinity chromatography techniques, which will be used to obtain definitive structural analyses of the sterol compounds formed by the enzyme reaction using different sterols substrates and allow detailed structural analysis of this unusual SAM:SMT enzyme protein.     

UDPG:Sterol glucosyltransferase in etiolated pea seedlings

Sterol UDPglucose glucosyltransferase was located predominantly in the axis tissue of etiolated pea seedlings. During the first 11 days of growth the activity reached a peak in the axis tissue after seven days. Centrifugation of tissue homogenates showed the cell fraction sedimenting between 13000 nd 25000 g to have the highest specific activity and also the bulk of the total activity. Sitosterol is the major free sterol of this fraction and cholesterol is a trace component. The composition of the aglycones of the isolated steryl glycosides shows cholesterol to be the major sterol. Although exhibiting no metal ion requirement, the enzyme is stimulated by Ca²⁺ and Mg²⁺, partially inhibited by EDTA and EGTA and completely inhibited by Zn²⁺. The membranous nature of the enzyme is manifested by its stimulation by the addition of phosphatidyl -ethanolamine, -choline and -serine. After brief treatment with phospholipases A, C and D, enzyme activity is partially lost. After phospholipase A treatment the activity may be completely restored by the addition of phosphatidyl ethanolamine but phosphatidyl-choline and -serine are without effect. After phospholipase C and D treatment, each phospholipid brings about a partial recovery of activity but phosphatidyl ethanolamine is again superior.     

Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana

Stevia rebaudiana leaves accumulate a mixture of at least eight different steviol glycosides. The pattern of glycosylation heavily influences the taste perception of these intensely sweet compounds. The majority of the glycosides are formed by four glucosylation reactions that start with steviol and end with rebaudioside A. The steps involve the addition of glucose to the C-13 hydroxyl of steviol, the transfer of glucose to the C-2' and C-3' of the 13-O-glucose and the addition of glucose to the hydroxyl of the C-4 carboxyl group. We used our collection of ESTs, an UDP-glucosyltransferase (UGT)-specific electronic probe and key word searches to identify candidate genes resident in our collection. Fifty-four expressed sequence tags (ESTs) belonging to 17 clusters were found using this procedure. We isolated full length cDNAs for 12 of the UGTs, cloned them into an expression vector, and produced recombinant enzymes in Escherichia coli. An in vitro glucosyltransferase activity enzyme assay was conducted using quercetin, kaempferol, steviol, steviolmonoside, steviolbioside, and stevioside as sugar acceptors, and (14)C-UDP-glucose as the donor. Thin layer chromatography was used to separate the products and three of the recombinant enzymes produced labelled products that co-migrated with known standards. HPLC and LC-ES/MS were then used to further define those reaction products. We determined that steviol UGTs behave in a regioselective manner and propose a modified pathway for the synthesis of rebaudioside A from steviol.     

Subcellular localization of udpg: Nuatigenin glucosyltransferase in oat leaves

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.     

Structural requirements for transformation of substrates by the S-adenosyl-L-methionine:delta 24(25)-sterol methyltransferase. Inhibition by analogs of the transition state coordinate.

Microsomes from sunflower seedlings were used to investigate the transition state coordinate for the C-24 methylation reaction that mediates phytosterol biosynthesis. They were then used to study structurally related cationic and uncharged compounds of the natural sterol substrate, which were designed to interfere with the reaction progress. The hypothetical reaction course is described to proceed through an Sn2 formation of an activated complex involving the initial production of a covalent structure with a dative bond (methyl from AdoMet attacks si-face of the 24,25-double bond of the sterol) and the secondary production of a series of high energy intermediates, the stabilization of which determines the final C-24 methylated product. Derivatives of lanosterol and cholesterol with a methyl, hydrogen, oxygen, or bromine atom introduced into the side chain and/or at C-3 in place of the natural nucleophile were studied as inhibitors that interfere with the formation of the hypothetical tertiary isopropylcarbinyl cation intermediate in the conversion of cycloartenal to 24(28)-methylene cycloartanol. The data indicate the most potent inhibitor is a sterol with an aziridine group attached to C-24(25), which mimics the bridged C-24(25) carbenium ion generated in the transition state, and the methyltransferase possesses two strategic sites: one that recognizes the proximal end of the sterol acting as a proton donor and the other that recognizes the distal end that acts as a proton acceptor. The best fit (binding/catalysis) involves a flat sterol (including substrate and inhibitor) with intact unsubstituted nucleophilic centers at C-3 and C-24 and a freely rotating side chain that can assume the pseudocyclic conformation.     

A collagen:glucosyltransferase at the surface of malignant fibroblasts

3T12 fibroblasts possess glucosyltransferases that catalyze the transfer of glucose from UDP-Glucose to galactosylhydroxylysyl residues on collagenous acceptors. The presence of the enzyme activity at the cell surface is indicated by the following findings: a) suspensions of intact cells, as well as intact cell monolayers, glucosylate gelatinized collagen b) glucose transfer is not due to UDP-Glucose hydrolysis and subsequent intracellular utilization of the free glucose c) experiments using cell suspensions with known proportions of broken cells indicate that the glucosyltransferase activity is attributable to intact cells and not to contamination by intracellular enzymes from broken cells. The Km value for UDP-Glucose is about 20 microM. The enzyme has a pronounced requirement for manganese, and shows highest activity between 2 and 10 mM. The optimal Mn2+ concentration for the intracellular gelatin:glucosyltransferase activity is more restricted (5 to 10 mM). Glucosyltransferase activity is strongly inhibited by diamide and N-ethylmaleimide (5 mM), suggesting that intact sulfhydryl residues present in the enzyme are essential.     

Phosphatidylcholine liposomes containing cholesterol analogues with side chains of various lengths

The effect of the length of the side chain of sterols on their interaction with phosphatidylcholine was studied by measuring the permeability properties of liposomes constituted with sterol analogues with side chains of various lengths. The sensitivities of liposomes constituted with these sterol analogues toward digitonin and polyene antibiotics were also examined.The effects of sterols on phase transition of phosphatidylcholine were examined by measuring their effects on permeability increase due to perturbation of phase equilibrium and by differential scanning calorimetry. An analogue with a short side chain, isopropyl (C-22), had a very similar effect to cholesterol in suppressing the permeability increase, suggesting that the full length of the side chain is not necessary for this effect.The permeability of egg yolk phosphatidylcholine at 42°C was suppressed as much by the analogue C-22 as by cholesterol. Androstene-3-β-ol, an analogue without a side chain, however, had little suppressive effect. Thus it is concluded that the condensing effect of sterol requires a side chain, but not the full length of side chain.Liposomes constituted with analogues having a side chain with more than 5 carbon atoms showed maximum reactivity with a polyene antibiotic, amphotericin B, whereas those constituted with analogues having a side chain with less than 4 carbon atoms showed weaker reactivity. These findings indicate that a side chain with more than 5 carbon atoms is essential for the maximum interaction of liposomes with amphotericin B. Unlike amphotericin B, filipin reacted almost equally well with liposomes containing C-22 and with those containing cholesterol. Thus the chain length of the side chain of sterol is less important for interaction of liposomes with filipin than for their interaction with amphotericin B.Liposomes containing analogues having a side chain with more than 6 carbon atoms showed maximum reactivity with digitonin. Thus for the maximum interaction of liposomes with digitonin, the side chain of sterol should be longer than 6 carbon atoms.