The substrate specificities of sunflower and soybean phospholipases D using transphosphatidylation reaction

<p>Abstract</p> <p>Background</p> <p>Phospholipase D (PLD) belongs to a lipolytic enzyme subclass which catalyzes the hydrolysis and transesterification of glycerophospholipids at the terminal phosphodiester bond.</p> <p>Results</p> <p>In this work, we have studied the substrate specificity of PLDs from germinating sunflower seeds and cultured-soybean cells, using their capacity of transphosphatidylation. In the presence of a nucleophilic acceptor, such as [¹⁴C]ethanol, PLD catalyzes the production of phosphatidyl-[¹⁴C]-ethanol. The resulting product is easily identified since it is well separated from the other lipids by thin-layer chromatography. The main advantage of this assay is that the phospholipid used as substrate does not need to be radiolabelled and thus allow us a large choice of polar heads and fatty acids. <it>In vitro</it>, we observed that sunflower and soybean cell PLD show the following decreasing order of specificity: phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol; while phosphatidylserine and phosphatidylinositol are utilized much less efficiently.</p> <p>Conclusions</p> <p>The substrate specificity is modulated by the fatty acid composition of the phosphatidylcholine used as well as by the presence of other charged phospholipids.</p

Functional Characterization of the N-Terminal C2 Domain from Arabidopsis thaliana Phospholipase Dα and Dβ

Most of plant phospholipases D (PLD) exhibit a C2-lipid binding domain of around 130 amino acid residues at their N-terminal region, involved in their Ca2+-dependent membrane binding. In this study, we expressed and partially purified catalytically active PLDα from Arabidopsis thaliana (AtPLDα) in the yeast Pichia pastoris. The N-terminal amino acid sequence of the recombinant AtPLDα was found to be NVEETIGV and thus to lack the first 35 amino acid belonging to the C2 domain, as found in other recombinant or plant purified PLDs. To investigate the impact of such a cleavage on the functionality of C2 domains, we expressed, in E. coli, purified, and refolded the mature-like form of the C2 domain of the AtPLDα along with its equivalent C2 domain of the AtPLDβ, for the sake of comparison. Using Förster Resonance Energy Transfer and dot-blot assays, both C2 domains were shown to bind phosphatidylglycerol in a Ca2+-independent manner while phosphatidic acid and phosphatidylserine binding were found to be enhanced in the presence of Ca2+. Amino acid sequence alignment and molecular modeling of both C2 domains with known C2 domain structures revealed the presence of a novel Ca2+-binding site within the C2 domain of AtPLDα

Lipases or esterases: does it really matter? Toward a new bio-physico-chemical classification.

International audienceCarboxylester hydrolases, commonly named esterases, consist of a large spectrum of enzymes defined by their ability to catalyze the hydrolysis of carboxylic ester bonds and are widely distributed among animals, plants, and microorganisms. Lipases are lipolytic enzymes which constitute a special class of carboxylic esterases capable of releasing long-chain fatty acids from natural water-insoluble carboxylic esters. However, up to now, several unsuccessful attempts aimed at differentiating "lipases" from "esterases" by using various criteria. These criteria were based on the first substrate used chronologically, primary sequence comparisons, some kinetic parameters, or some structural features.Lipids are biological compounds which, by definition, are insoluble in water. Taking into account this basic physico-chemical criterion, we primarily distinguish lipolytic esterases (L, acting on lipids) from nonlipolytic esterases (NL, not acting on lipids). In view of the biochemical data accumulated up to now, we proposed a new classification of esterases based on various criteria of physico-chemical, chemical, anatomical, or cellular nature. We believe that the present attempt matters scientifically for several reasons: (1) to help newcomers in the field, performing a few key experiments to figure out if a newly isolated esterase is lipolytic or not; (2) to clarify a debate between scientists in the field; and (3) to formulate questions which are relevant to the still unsolved problem of the structure-function relationships of esterases

Plant phospholipase D mining unravels new conserved residues important for catalytic activity

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Development of a Direct and Continuous Phospholipase D Assay Based on the Chelation-Enhanced Fluorescence Property of 8‑Hydroxyquinoline

Through its production of phosphatidic acid (PA), phospholipase D (PLD) is strongly involved in vesicular trafficking and cell signaling, making this enzyme an important therapeutic target. However, most PLD assays developed so far are either discontinuous or based on the indirect determination of choline released during PLD-catalyzed phosphatidylcholine hydrolysis, making its kinetic characterization difficult. We present here the development of a direct, specific, and continuous PLD assay that is based on the chelation-enhanced fluorescence property of 8-hydroxyquinoline (8HQ) following Ca²⁺ complexation with PLD-generated PA. The real-time fluorescence intensity from 8HQ/Ca²⁺/PA complexes can be converted to concentrations of product using a calibration curve, with a detection limit of 1.2 μM of PA on a microplate scale, thus allowing measurement of the PLD-catalyzed reaction rate parameters. Hence, this assay is well adapted for studying the substrate specificity of PLD, together with its kinetic parameters, using natural phospholipids with various headgroups. In addition, the assay was found to be effective in monitoring the competitive inhibition of PA formation in the production of phosphatidylalcohols following the addition of primary alcohols, such as ethanol, propan-1-ol, or butan-1-ol. Finally, this assay was validated using the purified recombinant <i>Vigna unguiculata</i> PLD, as well as the PLD from <i>Streptomyces chromofuscus</i>, cabbage, or peanuts, and no PA production could be detected using phospholipase A₁, phospholipase A₂, or phospholipase C, allowing for a reliable determination of PLD activity in crude protein extract samples. This easy to handle PLD assay constitutes, to our knowledge, the first direct and continuous PA determination method on a microplate scale

Phospholipases: An Overview

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Reassessing the Potential Activities of Plant CGI-58 Protein

International audienceComparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylgly-cerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvesti-gating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lyso-phosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombi-nant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed

Exploring the influence of phospholipid monolayer conformation and environmental conditions on the interfacial binding of Gibberella Zeae lipase

International audienceThe involvement of different parameters on Gibberella zeae lipase (GZEL) membrane binding were characterized by using monomolecular film technology and circular dichroism spectroscopy. Among four kinds of phospholipid monolayers, 1,2‑dimyristoyl‑sn‑glycero‑3‑phosphoethanolamine have the highest maximum insertion pressure (MIP) value. Comparing the GZEL adsorption to phosphatidylcholine monolayers with different acyl chains in sn-1 and sn-2 positions, the higher MIP values were found for 1,2‑dilauroyl‑sn‑glycero‑3‑phosphocholine. Significantly improvement between 1,2‑dioleoyl‑sn‑glycero‑3‑phosphocholine and 1,2‑distearoyl‑sn‑glycero‑3‑phosphocholine suggested that the presence of fatty acid unsaturation may affect protein adsorption by changing the chemical structure in each phospholipid. The MIP value was shown higher (48.6 mN m−1) at pH 5 and pH 6 (47.5 ± 1.9 mN m−1) but decreased significantly (34.2 mN m−1) at pH 9. This may indicate that the proportion of helices in the protein decreases with the alteration of the catalytic center, thus affecting the binding of the protein to its substrate. The MIP values obviously decreased with increasing salt ion concentration, suggesting that excessive salt ion concentration may destabilize the secondary and tertiary structures of the protein, thereby affecting the characteristics of its adsorption at the interfaces. Present studies improve our understanding on the protein-membrane interaction of this enzyme