Diacylglycerol Galactosyltransferase Research Articles

Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols.

A processive diacylglycerol glucosyltransferase has recently been identified from Bacillus subtilis [Jorasch, P., Wolter, F.P., Zähringer, U., and Heinz, E. (1998) Mol. Microbiol. 29, 419-430]. Now we report the cloning and characterization of two other genes coding for diacylglycerol glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana; only the S. aureus enzyme shows processivity similar to the B. subtilis enzyme. Both glycosyltransferases characterized in this work show unexpected acceptor specificities. We describe the isolation of the ugt106B1 gene (GenBank accession number Y14370) from the genomic DNA of S. aureus and the ugt81A1 cDNA (GenBank accession number AL031004) from A. thaliana by PCR. After cloning and expression of S. aureus Ugt106B1 in Escherichia coli, SDS/PAGE of total cell extracts showed strong expression of a protein having the predicted size of 44 kDa. Thin-layer chromatographic analysis of the lipids extracted from the transformed E. coli cells revealed several new glycolipids and phosphoglycolipids not present in the controls. These lipids were purified from lipid extracts of E. coli cells expressing the S. aureus gene and identified by NMR and mass spectrometry as 1, 2-diacyl-3-[O-beta-D-glucopyranosyl]-sn-glycerol, 1, 2-diacyl-3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyrano-+ ++syl] -sn-glycerol, 1, 2-diacyl-3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-( 1-->6)-O-beta-D-glucopyranosyl]-sn-glycerol, sn-3'-[O-beta-D-glucopyranosyl]-phosphatidylglycerol and sn-3'-[O-(6"'-O-acyl)-beta-D-glucopyranosyl-(1"'-->6")-O-beta-D-gluco pyranosyl]-sn-2'-acyl-phospha-tidylglycerol. A 1, 2-diacyl-3-[O-beta-D-galactopyranosyl]-sn-glycerol was isolated from extracts of E. coli cells expressing the ugt81A1 cDNA from A. thaliana. The enzymatic activities expected to catalyze the synthesis of these compounds were confirmed by in vitro assays with radioactive substrates. Experiments with several of the above described glycolipids as 14C-labeled sugar acceptors and unlabeled UDP-glucose as glucose donor, suggest that the ugt106B1 gene codes for a processive UDP-glucose:1, 2-diacylglycerol-3-beta-D-glucosyltransferase, whereas ugt81A1 codes for a nonprocessive diacylglycerol galactosyltransferase. As shown in additional assays with different lipophilic acceptors, both enzymes use diacylglycerol and ceramide, but Ugt106B1 also accepts glucosyl ceramide as well as cholesterol and cholesterol glucoside as sugar acceptors.

Uridine-diphospho-sulfoquinovose: diacylglycerol sulfoquinovosyltransferase activity is concentrated in the inner membrane of chloroplast envelopes

In experiments on the assembly of the sulfolipid sulfoquinovosyl diacylglycerol in envelope membranes of chloroplasts, UDP-sulfoquinovose (UDPS) was used with highest efficiency, and the corresponding enzyme, UDP-sulfoquinovose:diacylglycerol sulfoquinovosyltransferase, was partially characterized (E. Heinz et al., 1989, Eur J Biochem 184: 445–453). Here, we identified 35S- and 33P-labelled UDPS from various photosynthetically active organisms, suggesting that the sulfosugar nucleotide used for sulfolipid biosynthesis throughout the plant kingdom, including phototrophic bacteria, may indeed be UDPS. For attribution of the sulfolipid synthase to one of the two plastidial envelope membranes, these membranes were isolated from pea and spinach chloroplasts. The sulfoquinovosyltransferase was localized in the inner membrane of envelopes, which also contains the competing UDP-galactose:diacylglycerol galactosyltransferase. In contrast to the sulfoquinovosyltransferase, a substantial proportion of the galactosyltransferase was found in the outer membranes of envelopes from pea chloroplasts.

In Situ Incorporation of Fatty Acids into Lipids of the Outer and Inner Envelope Membranes of Pea Chloroplasts

When incubated with [1-(14)C]acetate and cofactors (ATP, Coenzyme A, sn-glycerol-3-phosphate, UDPgalactose, and NADH), intact chloroplasts synthesized fatty acids that were subsequently incorporated into most of the lipid classes. To study lipid synthesis at the chloroplast envelope membrane level, (14)C-labeled pea (Pisum sativum) chloroplasts were subfractionated using a single flotation gradient. The different envelope membrane fractions were characterized by their density, lipid and polypeptide composition, and the localization of enzymic activities (UDPgalactose-1,2 diacylglycerol galactosyltransferase, Mg(2+)-dependent ATPase). They were identified as very pure outer membranes (light fraction) and strongly enriched inner membranes (heavy fraction). A fraction of intermediate density, which probably contained double membranes, was also isolated. Labeled glycerolipids recovered in the inner envelope membrane were phosphatidic acid, phosphatidyl-glycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol. Their (14)C-fatty acid composition indicated that a biosynthetic pathway similar to the prokaryotic pathway present in cyanobacteria occurred in the inner membrane. In the outer membrane, phosphatidylcholine was the most labeled glycerolipid. Phosphatidic acid, phosphatidylglycerol, 1,2 diacylglycerol, and monogalactosyldiacylglycerol were also labeled. The (14)C-fatty acid composition of these lipids showed a higher proportion of oleate than palmitate. This labeling, different from that of the inner membrane, could result either from transacylation activities or from a biosynthetic pathway not yet described in pea and occurring partly in the outer chloroplast envelope membrane. This metabolism would work on an oleate-rich pool of fatty acids, possibly due to the export of oleate from chloroplast toward the extrachloroplastic medium. The respective roles of each membrane for chloroplast lipid synthesis are emphasized.

Enzymatic Characteristics of UDP‐sulfoquinovose: Diacylglycerol Sulfoquinovosyltransferase from Chloroplast Envelopes*

AbstractEnvelope membranes from chloroplasts contain UDP‐sulfoquinovose: diacylglycerol sulfoquinovosyltransferase which catalyses the final step in sulfolipid assembly. In situ produced diacylglycerol served as radioactive acceptor to measure enzymatic activity. With this assay, several enzymatic parameters were investigated. The enzyme, which has maximal activity at pH 7.5, was stimulated by magnesium ions due to a decrease of the Km for uridine 5′‐diphospho‐sulfoquinovose from 80 pM (no magnesium) to 10 μM (5 mM magnesium). This stimulation had a Km of 0.7 mM magnesium and may be relevant in light/dark modulation of the enzymatic activity. The lower efficiency of guanosine 5′‐diphospho‐sulfoquinovose observed before can be ascribed to a higher Km of this sugar nucleotide (400 μM). Under optimized and linearized conditions the sulfoquinovosyltransferase displayed about 10% of the activity of the UDP‐galactose: diacylglycerol galactosyltransferase which competes in the same membrane system for diacylglycerols. Addition of acidic lipids, such as sulfolipid and phosphatidylglycerol, to envelope membranes resulted in an inhibition of the sulfoquinovosyltransferase, whereas the galactosyltransferase was not affected. In vivo this may contribute to an adjustment of the sulfolipid proportion in plastid membranes. In contrast to the galactosyltransferase the sulfoquinovosyltransferase did not discriminate against the dipalmitoyl molecular species of diacylglycerol when offered together with the oleoyl‐palmitoyl species. Under conditions when oleoyl‐palmitoyl‐and dipalmitoyl‐diacylglycerols were synthesized with concurrent conversion to monogalactosyl and sulfoquinovosyl diacylglycerol, the sulfolipid was highly enriched in the fully saturated species. This may explain the occurrence of dipalmitoyl species in sulfolipids, as found in many plants.

Biosynthesis of Digalactosyldiacylglycerol in Plastids from 16:3 and 18:3 Plants

Intact chloroplasts isolated from leaves of eight species of 16:3 and 18:3 plants and chromoplasts isolated from Narcissus pseudonarcissus L. flowers synthesize galactose-labeled mono-, di-, and trigalactosyldiacylglycerol (MGDG, DGDG, and TGDG) when incubated with UDP-[6-(3)H]galactose. In all plastids, galactolipid synthesis, and especially synthesis of DGDG and TGDG, is reduced by treatment of the organelles with the nonpenetrating protease thermolysin. Envelope membranes isolated from thermolysin-treated chloroplasts of Spinacia oleracea L. (16:3 plant) and Pisum sativum L. (18:3 plant) or membranes isolated from thermolysin-treated chromoplasts are strongly reduced in galactolipid:galactolipid galactosyltransferase activity, but not with regard to UDP-Gal:diacylglycerol galactosyltransferase. For the intact plastids, this indicates that thermolysin treatment specifically blocks DGDG (and TGDG) synthesis, whereas MGDG synthesis is not affected. Neither in chloroplast nor in chromoplast membranes is DGDG synthesis stimulated by UDP-Gal. DGDG synthesis in S. oleracea chloroplasts is not stimulated by nucleoside 5'-diphospho digalactosides. Therefore, galactolipid:galactolipid galactosyltransferase is so far the only detectable enzyme synthesizing DGDG. These results conclusively suggest that the latter enzyme is located in the outer envelope membrane of different types of plastids and has a general function in DGDG synthesis, both in 16:3 and 18:3 plants.

UDPgalactose-independent synthesis of monogalactosyldiacylglycerol. An enzymatic activity of the spinach chloroplast envelope

Envelope membranes isolated from spinach chloroplasts synthesize monogalactosyldiacylglycerol (MGDG) from sn-1,2-diacylglycerol in the absence of UDPgalactose. The enzyme responsible galactosylates di[1- 14C]oleoylglycerol and diacyl[2- 3H]glycerol with rates comparable to the rate of UDPgalactose:di-acylglycerol galactosyltransferase (EC 2.4.1.46), if the latter enzyme is measured separately from other galactosyltransferase activities (Heemskerk J.W.M. et al. (1987) Biochim. Biophys. Acta 918, 189–203). The velocity of UDPgalactose-independent synthesis of MGDG increases considerably with the concentration of diacylglycerol, the maximal velocity being 550 nmol/mg envelope protein per h. A galactolipid, probably digalactosyldiacylglycerol (DGDG), serves as galactosyl donor. The reaction is not affected by the presence of UDP, Mg 2+ or EDTA. It is inhibited by treatment of the chloroplasts with the proteinase thermolysin, indicating a localization in the outer envelope membrane. Together, these results suggest that UDPgalactose-independent synthesis of MDGD is catalyzed by reversible action of galactolipid:galactolipid galactosyltransferase (EC 2.4.1.-). The enzymatic reaction may provide a separate pathway for the synthesis of MDGD in vivo, but can also be understood as a mechanism to maintain a low level of diacylglycerol in the envelope membrane or, alternatively, as a way to facilitate transfer of galactosyl groups between galactolipid molecules.

Lipid requirement and kinetic studies of solubilized UDP-galactose:diacylglycerol galactosyltransferase activity from spinach chloroplast envelope membranes

We have demonstrated a lipid requirement for the UDPgalactose:1,2-diacylglycerol 3-beta-D-galactosyl-transferase (or monogalactosyldiacylglycerol synthase; EC 2.4.1.46), an enzyme involved in the biosynthesis of monogalactosyldiacylglycerol, solubilized from chloroplast envelope membranes and partially purified by hydroxyapatite chromatography. The enzyme fraction was highly delipidated (<0.1 mg of lipid per mg of protein), and addition of lipids extracted from chloroplast membranes was necessary to reveal the activity. Acidic glycerolipids, and especially phosphatidylglycerol, were the best activators of the enzyme. The preparation of a delipidated enzyme fraction and the development of optimal assay conditions were prerequisites for the determination of the kinetic parameters for the hydrophobic substrate of the enzyme, diacylglycerol. In addition, we have demonstrated the existence of two substrate-binding sites: a hydrophobic one for diacylglycerol and a hydrophilic one for UDP-galactose.

Synthesis of Mono- and Digalactosyldiacylglycerol in Isolated Spinach Chloroplasts

Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose-labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U-(14)C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-(14)C]acetate or sn-[U-(14)C]glycerol-3-P in the presence of Mg(2+) and unlabeled UDP-Gal resulted in high (14)C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase.

Solubilization and partial purification of UDP-galactose:diacylglycerol galactosyltransferase activity from spinach chloroplast envelope

We have developed procedures to solubilize the envelope UDP-galactose:diacylglycerol galactosyltransferase activity and to assay this enzyme after solubilization with a zwitterionic non-denaturing detergent (CHAPS) and fractionation. From solubilized envelope membranes isolated from intact spinach chloroplasts, we were able to prepare by chromatography on hydroxyapatite a fraction enriched (6–7-fold) in this enzyme responsible for monogalactosyldiacylglycerol synthesis.

Separation of chloroplast polar lipids and measurement of galactolipid metabolism by high-performance liquid chromatography

Procedures are described for the separation of polar lipids from plant chloroplasts by high-performance liquid chromatography, using a polar-modified silica column. Glycolipids and phospholipids were eluted with a gradient of 2-propanol/ n-hexane (80:55, v v ) and 2-propanol/ n-hexane/water/methanol (80:55:15:10, v v ). The lipids were detected by uv absorbance at 202 nm. Diacylglycerol and mono-, di-, and trigalactosyldiacylglycerol and phosphatidylcholine were separated on a LiChrosorb NH 2 column (7-μm particles, Merck, FRG), but acidic lipids were retained. These lipids could be quantified from their 202-nm absorbance recording. The absorption coefficients obtained depended on the mean number of double bonds in the different lipid classes. The separation was applied for a rapid monitoring of the lipid composition in thylakoids and in fractionated inner and outer envelopes. The activities of galactosyltransferases involved in galactolipid metabolism, UDPGal:diacylglycerol galactosyltransferase and galactolipid:galactolipid galactosyltransferase, could be measured quantitatively in specific assays for both enzymes.

Spinach chloroplasts: localization of enzymes involved in galactolipid metabolism

A shortened procedure for the separation of spinach chloroplast envelope membranes is presented, resulting in high yields of enriched inner and outer envelopes. Fractionated envelopes were tested for activities in galactolipid synthesis by measuring incorporation of UDPGal and by using a specific assay for galactolipid: galactolipid galactosyltransferase (GGGT); additionally, acyl-CoA synthetase activities were tested. UDPGal incorporation and GGGT were found predominantly in enriched inner envelopes, acyl-CoA synthetase was predominantly in enriched outer envelopes. These observations point to a localization of both UPDGal: diacylglycerol galactosyltransferase (UDGT) and GGGT in the inner membrane. Alternatively, chloroplasts were incubated with [ 14C]acetate under conditions permitting fatty acid synthesis in the presence and absence of UDP[ 3H]Gal. After incubation, envelope fractions were isolated and analyzed for labeled lipids. Analysis agreed with localization of UDGT in the inner envelope. However, labeled products of GGGT were found both in enriched inner and outer envelopes. Furthermore, inactivation of GGGT by the proteinase thermolysin indicates a location on the cytosolic face of the chloroplast. These apparently contradictory results concerning GGGT might be explained by the involvement of contact sites between inner and outer envelopes or by lipid transformation during membrane fractionation.

Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. II. Biochemical characterization.

In the previous paper (Block, M. A., Dorne, A.-J., Joyard, J., and Douce, R. (1983) J. Biol. Chem. 258, 13273-13280), we have described a method for the separation of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. The two envelope membranes have a different weight ratio of acyl lipid to protein (2.5-3 for the outer envelope membrane and 0.8-1 for the inner envelope membrane). The two membranes also differ in their polar lipid composition. However, in order to prevent the functioning of the galactolipid:galactolipid galactosyltransferase during the course of envelope membrane separation, we have analyzed the polar lipid composition of each envelope membrane after thermolysin treatment of the intact chloroplasts. The outer envelope membrane is characterized by the presence of high amounts of phosphatidylcholine and digalactosyldiacylglycerol whereas the inner envelope membrane has a polar lipid composition almost identical with that of the thykaloids. No phosphatidylethanolamine or cardiolipin could be detected in either envelope membranes, thus demonstrating that the envelope membranes, and especially the outer membrane, do not resemble extrachloroplastic membranes. No striking differences were found in the fatty acid composition of the polar lipids from either the outer or the inner envelope membrane. The two envelope membranes also differ in their carotenoid composition. Among the different enzymatic activities associated with the chloroplast envelope, we have shown that the Mg2+-dependent ATPase, the UDP-Gal:diacylglycerol galactosyltransferase, the phosphatidic acid phosphatase, and the acyl-CoA thioesterase are associated with the inner envelope from spinach chloroplasts whereas the acyl-CoA synthetase is located on the outer envelope membrane.

Turnover of galactolipids incorporated into chloroplast envelopes: An assay for galactolipid: Galactolipid galactosyltransferase

An assay for the measurement of galactolipid:galactolipid galactosyltransferase activity in chloroplast envelopes is presented. For this assay, [ 14C]galactose-labeled monogalactosyl diacylglycerol and sodium desoxycholate (in the molar ratio 1:10) are sonicated together, and the resulting suspension is resonicated with the chloroplast envelopes (in the molar ratio total galactolipids/sodium desoxycholate = 0.6). The incorporated monogalactosyl diacylglycerol is readily converted into di-, tri-, tetra- and, presumably, pentagalactosyl diacylglycerol. The galactosyltransferase activity shows a broad pH optimum of 5.9–7.0, it is still active at temperatures as low as 4°C and is stimulated by Mg 2+ and Mn 2+. The detergent-modified envelopes show a reduced incorporation of UDP[ 3H]Gal, which results, however, in a quite normal pattern of 3H-Iabeled galactolipids. The rate of conversion of the incorporated [ 14C]monogalactosyl diacylglycerol was not influenced by the presence or absence of UDP[ 3H]Gal. Evidence is supplied for the absence of UDPgalactose:monogalactosyl diacylglycerol galactosyltransferase activity in our system. Incorporation of [ 14C)digalactosyl diacylglycerol into the envelopes yields labeled tri- and tetragalactosyl diacylglycerol and also monogalactosyl diacylglycerol. The possibility of reversed galactolipid: galactolipid galactosyltransferase activity is discussed. Incorporated monogalactosyl monoacylglycerol is converted partly into a compound which is presumably digalactosyl monoacylglycerol.

Lipids and Enzymatic Activities in Vacuolar Membranes Isolated via Protoplasts from Oat Primary Leaves

Vacuoles were released from oat (Avena sativa) mesophyll protoplasts and purified by sedi­mentation and flotation. Disruption of isolated vacuoles followed by density gradient centri­fugation gave two membrane bands which after combination were further purified on sucrose gradients. A significant contamination by microbodies, thylakoids, mitochondria, endoplasmic reticulum and Golgi membranes can be excluded, whereas markers for plasma membrane and chloroplast envelope were present in the final membrane preparation. In the purified membrane fraction the following enzymatic activities were detected: NADH- cytochrome C reductase E.C. 1.6.2.2, ATPase E.C. 3.6.1.3. UDPG: sterol glucosyltransferase, UDP-Gal: diacylglycerol galactosyltransferase E.C. 2.4.1.46, glucan synthetase II, CDP-choline: diacylglycerol phosphocholinetransferase E.C. 2.7.8.2, formation of acylgalactosyl diacylglycerol and acyl-CoA thioesterase E.C. 3.1.2.2. None of these can be considered to be specific for the tonoplast. Acid phosphatase E.C. 3.1.3.2 was present in the cell sap. The vacuolar membranes contain phospholipids and glycolipids of the most complex composi­tion found so far in a membrane system isolated from mesophyll protoplasts. About half of the glycolipids were accounted for by glycosyl diacylglycerols usually considered to be confined to plastids. Steryl glycosides and acyl steryl glycosides were other prominent glycolipids. A cerebroside was the predominating lipid component of this membrane preparation.

Galactosyltransferases Involved in Galactolipid Biosynthesis Are Located in the Outer Membrane of Pea Chloroplast Envelopes

The galactosylation steps in the biosynthesis of galactolipids involve two different enzymes; a UDP-Gal:diacylglycerol galactosyltransferase and a galactolipid:galactolipid galactosyltransferase. Previous localization studies have shown that in spinach these enzymes are located in the chloroplast envelope. Our results with peas (Pisum sativum var Laxton's Progress No. 9) confirm these results and extend the localization by providing evidence that the galactosyltransferases are in the outer membrane of the envelope. The specific activity of UDP-Gal:diacylglycerol galactosyltransferase in outer membrane preparations was 6 to 10 times greater than that exhibited by inner membrane preparations. In addition, using quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was possible to show that the UDP-Gal:diacylglycerol galactosyltransferase activity associated with inner membrane preparations could be accounted for by outer membrane contamination. It is concluded from these results that this enzyme is located predominantly, if not exclusively, in the outer membrane of the envelope. An analysis of the galactolipid products synthesized by the highly purified outer membrane showed that the galactolipid:galactolipid galactosyltransferase is also present, suggesting that this enzyme is also an outer membrane enzyme. The implication of these results is that the final assembly of galactolipids is carried out on the outer membrane of the chloroplast envelope.

Galactolipid synthesis in chromoplasts in vitro

Isolated chromoplasts from Narcissus pseudonarcissus flowers contain: a fatty acid synthesizing system; acyl-CoA synthetase (EC 6.2.1.3); glycero-phosphate acyltransferase (EC 2.3.1.15); acylglycero-phosphate acyltransferase; phosphatidate phosphatase (EC 3.1.3.4); diacylglycerol galactosyltransferase (EC 2.4.1.46); and diacylgalactosylglycerol galactosyltransferase, i.e. all enzymatic activities necessary for the synthesis of diacylgalactosylglycerol and diacylgalabiosylglycerol from acetate, HCO - (3) , sn-glycerol 3-phosphate, and UDP-D-galactose. Diacylgalactosylglycerol and diacylgalabiosylglycerol, however, are synthesized from these precursors to only a very low extent in an in vitro system. This is attributed to a specificity of diacylglycerol galactosyltransferase for highly unsaturated diacylglycerols. Specificities of acyltransferase reactions were also found.