植物GPATs基因研究进展

甘油-3-磷酸酰基转移酶(Glycerol-3-phosphate acyltransferase, GPAT)是三酰甘油(Triacylglycerol, TAG)生物合成的限速酶, 催化TAG生物合成的起始步骤。GPATs主要负责将脂肪酰基从酰基-酰基载体蛋白(acyl-ACP)或酰基辅酶A(acyl-CoA)上转移到甘油-3-磷酸的(Glycerol-3-phosphate, G3P) sn-1位置上。有些成员还具有sn-2酰基转移活性。目前已经在多种植物中克隆得到了GPAT基因。这些GPAT基因编码的酶主要分为三类, 它们在细胞中分别定位于质体、线粒体和内质网上。这些酶参与三酰甘油、几丁质和软木脂等多种脂质的生物合成, 在植物的生长发育中发挥着非常重要的作用。文章介绍了植物GPAT基因的染色体定位和基因结构以及GPAT酶的亚细胞定位、sn-2酰基转移特异性、GPAT酶的底物选择性及其生理功能的最新研究进展。     

sn-Glycerol-3-phosphate transport in Salmonella typhimurium

Salmonella typhimurium contains a transport system for sn-glycerol-3-phosphate that is inducible by growth on glycerol and sn-glycerol-3-phosphate. In fully induced cells, the system exhibited an apparent Km of 50 microM and a Vmax of 2.2 nmol/min . 10(8) cells. The corresponding system in Escherichia coli exhibits, under comparable conditions, a Km of 14 microM and a Vmax of 2.2 nmol/min . 10(8) cells. Transport-defective mutants were isolated by selecting for resistance against the antibiotic fosfomycin. They mapped in glpT at 47 min in the S. typhimurium linkage map, 37% cotransducible with gyrA. In addition to the glpT-dependent system, S. typhimurium LT2 contains, like E. coli, a second, ugp-dependent transport system for sn-glycerol-3-phosphate that was derepressed by phosphate starvation. A S. typhimurium DNA bank containing EcoRI restriction fragments in phage lambda gt7 was used to clone the glpT gene in E. coli. Lysogens that were fully active in the transport of sn-glycerol-3-phosphate with a Km of 33 microM and a Vmax of 2.0 nmol/min . 10(8) cells were isolated in a delta glpT mutant of E. coli. The EcoRI fragment harboring glpT was 3.5 kilobases long and carried only part of glpQ, a gene distal to glpT but on the same operon. The fragment was subcloned in multicopy plasmid pACYC184. Strains carrying this hybrid plasmid produced large amounts of cytoplasmic membrane protein with an apparent molecular weight of 33,000, which was identified as the sn-glycerol-3-phosphate permease. Its properties were similar to the corresponding E. coli permease. The presence of the multicopy glpT hybrid plasmid had a strong influence on the synthesis or assembly of other cell envelope proteins of E. coli. For instance, the periplasmic ribose-binding protein was nearly absent. On the other hand, the quantity of an unidentified E. coli outer membrane protein usually present only in small amounts increased.     

sn-Glycerol-3-phosphate transacylase activity in Euglena gracilis organelles

sn-Glycerol-3-phosphate transacylase activity was demonstrated in Euglena mitochondria, chloroplasts, and microsomes. There was no activity in the 100,000g 1-h supernatant. Exposure of each of the isolated organelles to 1 × 10^?4% Triton X-100 resulted in release of substantial quantities of transacylase activity into the 100,000g supernatant. Products formed by catalysis by the membrane-bound transacylases were heterogenous, while those resulting from catalysis by the extracted enzymes were practically all lysophosphatidate.     

sn-Glycerol-3-phosphate acyltransferase in a particulate fraction from maturing safflower seeds

The properties of the acyl-CoA:sn-glycerol-3-phosphate O-acyltransferase in a 20,000g particulate fraction from maturing safflower seeds were investigated. The optimum pH of the reaction was 7.2. The apparent Km for glycerophosphate was 0.54 mM. Only monoacylglycerophosphate was accumulated in the particulate fraction under normal conditions. Position 1 of glycerophosphate was exclusively esterified with either palmitoyl-CoA or linoleoyl-CoA as acyl donor, while 2-acylglycerophosphate was the minor product. The specificity and selectivity of the acyltransferase for acyl-CoA were broad and somewhat affected by temperature. The concentration of glycerophosphate did not affect the selectivity. These observations suggested that the fatty acid composition of position 1 of safflower triacylglycerol must primarily depend on the composition of the acyl-CoA pool in the site of synthesis, and that growth temperature and the acyl-CoA selectivity of the glycerophosphate acyltransferase may be rather minor factors regarding regulation of the fatty acid composition of position 1 in triacylglycerol.     

Purification and cDNA sequencing of an oleate-selective acyl-ACP:sn-glycerol-3-phosphate acyltransferase from pea chloroplasts

The soluble acyl-ACP:sn-glycerol-3-phosphate acyltransferase from chloroplasts of chilling-sensitive and -resistant plants differ in their fatty acid selectivity. Enzymes from resistant plants discriminate against non-fluid palmitic acid and select oleic acid whereas the acyltransferase from sensitive plants accepts both fatty acids. To use this difference for improving plant chilling resistance by biotechnology the gene for an oleate-selective enzyme is required. Therefore, the oleate-selective enzyme from pea seedlings was purified to apparent homogeneity. Tryptic peptides of internal origin were sequenced. Polyclonal antibodies raised in rabbits were used for an immunological screening of a pea leaf cDNA expression library in gt11. A positive clone of 1800 bp was selected showing an open reading frame which codes for 457 amino acids. The deduced amino acid sequence coincides perfectly with the tryptic sequences. A tentative assignment of the processing site was made which divides the preprotein into a mature protein of 41 kDa in accordance with experimental findings and a transit peptide of 88 amino acids. At present the comparison between a selective (pea) and an unselective (squash) acyltransferase sequence does not provide a clue for recognizing the structural differences resulting in different selectivities.     

Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn-Glycerol-1-phosphate Dehydrogenase

One of the most critical events in the origins of cellular life was the development of lipid membranes. Archaea use isoprenoid chains linked via ether bonds to sn-glycerol 1-phosphate (G1P), whereas bacteria and eukaryotes use fatty acids attached via ester bonds to enantiomeric sn-glycerol 3-phosphate. NAD(P)H-dependent G1P dehydrogenase (G1PDH) forms G1P and has been proposed to have played a crucial role in the speciation of the Archaea. We present here, to our knowledge, the first structures of archaeal G1PDH from the hyperthermophilic methanogen Methanocaldococcus jannaschii with bound substrate dihydroxyacetone phosphate, product G1P, NADPH, and Zn²⁺ cofactor. We also biochemically characterized the enzyme with respect to pH optimum, cation specificity, and kinetic parameters for dihydroxyacetone phosphate and NAD(P)H. The structures provide key evidence for the reaction mechanism in the stereospecific addition for the NAD(P)H-based pro-R hydrogen transfer and the coordination of the Zn²⁺ cofactor during catalysis. Structure-based phylogenetic analyses also provide insight into the origins of G1PDH.     

Characterization of the diversification of phospholipid:diacylglycerol acyltransferases in the green lineage

Triacylglycerols have important physiological roles in photosynthetic organisms, and are widely used as food, feed and industrial materials in our daily life. Phospholipid:diacylglycerol acyltransferase (PDAT) is the pivotal enzyme catalyzing the acyl‐CoA‐independent biosynthesis of triacylglycerols, which is unique in plants, algae and fungi, but not in animals, and has essential functions in plant and algal growth, development and stress responses. Currently, this enzyme has yet to be examined in an evolutionary context at the level of the green lineage. Some fundamental questions remain unanswered, such as how PDATs evolved in photosynthetic organisms and whether the evolution of terrestrial plant PDATs from a lineage of charophyte green algae diverges in enzyme function. As such, we used molecular evolutionary analysis and biochemical assays to address these questions. Our results indicated that PDAT underwent divergent evolution in the green lineage: PDATs exist in a wide range of plants and algae, but not in cyanobacteria. Although PDATs exhibit the conservation of several features, phylogenetic and selection‐pressure analyses revealed that overall they evolved to be highly divergent, driven by different selection constraints. Positive selection, as one major driving force, may have resulted in enzymes with a higher functional importance in land plants than green algae. Further structural and mutagenesis analyses demonstrated that some amino acid sites under positive selection are critically important to PDAT structure and function, and may be central in lecithin:cholesterol acyltransferase family enzymes in general.     

Expression and regulation of 1-acyl-sn-glycerol- 3-phosphate acyltransferases in the epidermis

Phospholipids are a major class of lipids in epidermis, where they serve as a source of free fatty acids that are important for the maintenance of epidermal permeability barrier function. The phospholipid biosynthetic enzyme, 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids. We identified an expression pattern of AGPAT isoforms that is unique to epidermis, with relatively high constitutive expression of mouse AGPAT (mAGPAT) 3, 4, and 5 but low constitutive expression of mAGPAT 1 and 2. Localization studies indicate that all five isoforms of AGPAT were expressed in all nucleated layers of epidermis. Furthermore, rat AGPAT 2 and 5 mRNAs increased in parallel with both an increase in enzyme activity and permeability barrier formation late in rat epidermal development. Moreover, after two methods of acute permeability barrier disruption, mAGPAT 1, 2, and 3 mRNA levels increased rapidly and were sustained for at least 24 h. In parallel with the increase in mRNA levels, an increase in AGPAT activity also occurred. Because upregulation of mAGPAT mRNAs after tape-stripping could be partially reversed by artificial barrier restoration by occlusion, these studies suggest that an increase in the expression of AGPATs is linked to barrier requirements.     

Glycerol-3-phosphate acyltransferases: Rate limiting enzymes of triacylglycerol biosynthesis

Four homologous isoforms of glycerol-3-phosphate acyltransferase (GPAT), each the product of a separate gene, catalyze the synthesis of lysophosphatidic acid from glycerol-3-phosphate and long-chain acyl-CoA. This step initiates the synthesis of all the glycerolipids and evidence from gain-of-function and loss-of-function studies in mice and in cell culture strongly suggests that each isoform contributes to the synthesis of triacylglycerol. Much work remains to fully delineate the regulation of each GPAT isoform and its individual role in triacylglycerol synthesis.     

LTP3 contributes to disease susceptibility in Arabidopsis by enhancing abscisic acid (ABA) biosynthesis

Several plant lipid transfer proteins (LTPs) act positively in plant disease resistance. Here, we show that LTP3 (At5g59320), a pathogen and abscisic acid (ABA)‐induced gene, negatively regulates plant immunity in Arabidopsis. The overexpression of LTP3 (LTP3‐OX) led to an enhanced susceptibility to virulent bacteria and compromised resistance to avirulent bacteria. On infection of LTP3‐OX plants with Pseudomonas syringae pv. tomato, genes involved in ABA biosynthesis, NCED3 and AAO3, were highly induced, whereas salicylic acid (SA)‐related genes, ICS1 and PR1, were down‐regulated. Accordingly, in LTP3‐OX plants, we observed increased ABA levels and decreased SA levels relative to the wild‐type. We also showed that the LTP3 overexpression‐mediated enhanced susceptibility was partially dependent on AAO3. Interestingly, loss of function of LTP3 (ltp3‐1) did not affect ABA pathways, but resulted in PR1 gene induction and elevated SA levels, suggesting that LTP3 can negatively regulate SA in an ABA‐independent manner. However, a double mutant consisting of ltp3‐1 and silent LTP4 (ltp3/ltp4) showed reduced susceptibility to Pseudomonas and down‐regulation of ABA biosynthesis genes, suggesting that LTP3 acts in a redundant manner with its closest homologue LTP4 by modulating the ABA pathway. Taken together, our data show that LTP3 is a novel negative regulator of plant immunity which acts through the manipulation of the ABA–SA balance.     

sn-Glycerol-3-phosphate dehydrogenase and its interaction with nitrate reductase in wild-type and hem mutant strains of Staphylococcus aureus

Staphylococcus aureus has membrane-associated sn-glycerol-3-phosphate dehydrogenase activity that is strongly activated by detergents. The enzyme can be measured spectrophotometrically in intact cells in assay systems containing lauryldimethylamine oxide (Ammonyx LO). The dehydrogenase activity was located exclusively in the membrane fraction of cells grown with glycerol under aerobic conditions or under anaerobic conditions with the addition of nitrate; there was no evidence of multiple forms. Development of sn-glycerol-3-phosphate dehydrogenase activity was studied with suspensions of cells grown previously under semianaerobic conditions with glucose and nitrate. The wild-type strain rapidly formed the enzyme when incubated with glycerol under aerobic conditions or under semianaerobic conditions in the presence of nitrate. Under similar conditions, suspensions of hem mutant H-14 required the addition of hemin. Induction of the enzyme was strongly repressed by glucose with both organisms. A procedure was established to obtain cells of mutant H-14 with sn-glycerol-3-phosphate dehydrogenase and nitrate reductase activities, but which could not link the systems unless supplemented with hemin. The coupled activity could also be reconstructed in vitro by the addition of hemin to the depleted membranes.     

Hepatic sn-glycerol-3-phosphate acyltransferases: effect of monoacylglycerol analogs on mitochondrial and microsomal activities

The regulation of cellular diacylglycerol levels may have important consequences for protein kinase C activity. Because monoacylglycerols were said to inhibit the committed step of glycerolipid synthesis, the sn-glycerol-3-P acyltransferase (glycerol-P acyltransferase), we determined (1) whether both the mitochondrial and the microsomal glycerol-P acyltransferase isoenzymes were inhibited by 1- and 2-mono-18:1-glycerols, and their ether and amide analogs and (2) what the mechanism of inhibition was. 1- and 2-mono-18:1-glycerols, their ether and amide analogs, and 1-mono-18:1-glycerol 3-phosphate were all competitive inhibitors of the microsomal glycerol-P acyltransferase activity. The relative Ki values suggested that inhibition was strongest with the radyl group at the sn-1 position and that an oxygen bond is important at the sn-1 position. Although the monoacyl- and monoalkylglycerols were also competitive inhibitors of the mitochondrial glycerol-P acyltransferase, neither of the amide analogs was an inhibitor, suggesting that an oxygen bond is essential at both the sn-1 and sn-2 positions. Because monoradylglycerols inhibit several enzyme activities that contribute to the biosynthesis or the metabolism of diacylglycerol, these inhibitors may function within cells in part to regulate cellular diacylglycerol levels.     

Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase

The methylerythritol 4-phosphate (MEP) pathway synthesizes the precursors for an astonishing diversity of plastid isoprenoids, including the major photosynthetic pigments chlorophylls and carotenoids. Since the identification of the first two enzymes of the pathway, deoxyxylulose 5-phoshate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), they both were proposed as potential control points. Increased DXS activity has been shown to up-regulate the production of plastid isoprenoids in all systems tested, but the relative contribution of DXR to the supply of isoprenoid precursors is less clear. In this work, we have generated transgenic Arabidopsis thaliana plants with altered DXS and DXR enzyme levels, as estimated from their resistance to clomazone and fosmidomycin, respectively. The down-regulation of DXR resulted in variegation, reduced pigmentation and defects in chloroplast development, whereas DXR-overexpressing lines showed an increased accumulation of MEP- derived plastid isoprenoids such as chlorophylls, carotenoids, and taxadiene in transgenic plants engineered to produce this non-native isoprenoid. Changes in DXR levels in transgenic plants did not result in changes in␣DXS gene expression or enzyme accumulation, confirming that the observed effects on plastid isoprenoid levels in DXR-overexpressing lines were not an indirect consequence of altering DXS levels. The results indicate that the biosynthesis of MEP (the first committed intermediate of the pathway) limits the production of downstream isoprenoids in Arabidopsis chloroplasts, supporting a role for DXR in the control of the metabolic flux through the MEP pathway.     

The C-terminal region of mitochondrial glycerol-3-phosphate acyltransferase-1 interacts with the active site region and is required for activity

Glycerol phosphate acyltransferase (GPAT) catalyzes the formation of 1-acyl-sn-glycerol-3-phosphate from glycerol-3-phosphate and long chain fatty acyl-CoA substrates. We previously determined the topography of the mitochondrial GPAT1 isoform (mtGPAT1, 828 amino acids). mtGPAT1 has two transmembrane domains (TMDs) (aa 472-493 and aa 576-592) with both the N- and C-termini facing the cytosol and a loop (aa 494-575) facing the intermembrane space. Alignment of amino acid sequences from mtGPAT1 and other acyltransferases and site directed mutagenesis studies have demonstrated that the active site of the enzyme resides in the N-terminal domain of the protein. In this study, we sequentially truncated the C-terminal domain and characterized the properties of the resulting mutants expressed in CHO cells. Although the mutants were overexpressed, none of them conferred GPAT activity. The loss of activity was not due to the miss-targeting of the proteins since immunofluorescence experiments demonstrated their mitochondrial localization. Instead, chemical crosslinking and protein cleavage studies demonstrated that the N- and C-termini of the protein interact. These results suggest that the C-terminal domain is necessary for mtGPAT1 activity, and probably contributes to catalysis or substrate binding.     

Analysis of Arabidopsis thaliana transgenic plants transformed with CER2 and CER3 genes in sense and antisense orientations

 A number of genes involved in the biosynthesis of the epicuticular wax (EW) of Arabidopsis thaliana have recently been isolated through genetic approaches. In view of the evidence in favor of the importance of EW compounds in the adaptation of higher plants to a number of physiological and ecological stresses, we have used clones of some of these genes to genetically engineer constructs with which to manipulate EW biosynthesis in transgenic A. thaliana plants. All our constructs were placed under the control of the near constitutive CaMV 35S promoter. We were able to complement mutant plants with the construction in the sense orientation as well as induce phenocopies of the eceriferum phenotype by transforming wild-type plants with both the sense and antisense constructs. We observed reduced fertility in the wild-type plants transformed with the 35SCER3sense or 35SCER3antisense constructs but not in those transformed with the 35SCER2sense or 35SCER2antisense constructs. Received: 28 December 1997 / Accepted: 31 March 1998     

Arabidopsis phosphatidylglycerophosphate phosphatase 1 involved in phosphatidylglycerol biosynthesis and photosynthetic function

Phosphatidylglycerol (PG) is an indispensable lipid constituent of photosynthetic membranes, whose function is essential in photosynthetic activity. In higher plants, the biological function of the last step of PG biosynthesis remains elusive because an enzyme catalyzing this reaction step, namely phosphatidylglycerophosphate phosphatase (PGPP), has been a missing piece in the entire glycerolipid metabolic map. Here, we report the identification and characterization of AtPGPP1 encoding a PGPP in Arabidopsis thaliana. Heterologous expression of AtPGPP1 in yeast Δgep4 complemented growth phenotype and PG‐producing activity, suggesting that AtPGPP1 encodes a functional PGPP. The GUS reporter assay showed that AtPGPP1 was preferentially expressed in hypocotyl, vasculatures, trichomes, guard cells, and stigmas. A subcellular localization study with GFP reporter indicated that AtPGPP1 is mainly localized at chloroplasts. A T‐DNA‐tagged knockout mutant of AtPGPP1, designated pgpp1‐1, showed pale green phenotype with reduced PG and chlorophyll contents but no defect in embryo development. In the pgpp1‐1 mutant, ultrastructure of plastids indicated defective development of chloroplasts and measurement of photosynthetic parameters showed impaired photosynthetic activity. These results suggest that AtPGPP1 is a primary plastidic PGPP required for PG biosynthesis and photosynthetic function in Arabidopsis.     

Higher plants express 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Abstract. The enzymatic activity of 3-deoxy- D-manno -octulosonate 8-phosphate (KDOP) synthase was detected in eight diverse plant species, thus providing enzymological data consistent with recent reports of the presence of 3-deoxy- D-manno -octulosonate in plant cell walls. KDOP synthase from spinach was partially purified and characterized. It possessed weak activity as 3-deoxy- D-arabino -heptulosonate 7-phosphate (DAHP) synthase. In the presence of phosphoenolpyruvate, which conferred dramatic thermostability, KDOP synthase had a catalytic temperature optimum of about 53°C. The pH optimum was 6.2, and divalent cations were neither stimulatory nor required for activity. The Km values for arabinose 5-P and phosphoenolpyruvate were 0.27 mol m⁻³ and about 35 mmol m⁻³, respectively. The kinetics of periodate oxidation of KDOP formed by spinach KDOP synthase indicate that the same stereochemical configuration exists as with bacterial KDOP. The possibility that an unregulated species of DAHP synthase found in some bacteria might in fact be a KDOP synthase exhibiting substrate ambiguity of the type seen in higher plants was examined. However, the DAHP synthase isozyme, DS-O, from Acinetobacter calcoaceticus was found to be specific for erythrose 4-P. The KDOP synthase of Acinetobacter calcoaceticus was also found to be specific for arabinose 5-P.     

Changes in the activities of dihydroxyacetone phosphate and glycerol-3-phosphate acyltransferases in rat liver under various conditions

Activities of enzymes relating to the acyl dihydroxyacetone phosphate (acyl DHAP) pathway were determined in rat liver under conditions known to elevate the peroxisomal β-oxidation activity. In fasted and streptozotocin-induced diabetic rats, DHAP acyltransferase activity showed a small but significant increase, though the activities of glycerol-3-phosphate (GP) acyltransferase and alkyl DHAP synthase were not changed. After 2 weeks, feeding of 20% partially hydrogenated marine oil, the activity of DHAP acyltransferase also increased to 140% of the control. The feeding of 0.25% clofibrate and 2% di(2-ethylhexyl)phthalate (DEHP) increased the activities of both DHAP and GP acyltransferases by 2- to 3-fold, whereas alkyl DHAP synthase activity decreased under the same conditions. A fractionation study showed that the increases in the activities of DHAP acyltransferase and acyl /alkyl DHAP reductase in the liver of rats treated with DEHP occurred mainly in peroxisomes and microsomes, respectively. The phospholipid contents per mg protein of the isolated hepatic peroxisomes from rats were as follows (percent of the control): fasting, 62%; diabetic, 69%; high fat-diet, 89%; clofibrate-treated, 126%; DEHP-treated, 119%. These results suggest that glycerophospholipid metabolism might also be controlled by peroxisomal enzymes under physiological and pathological conditions.