Phospholipase D from Streptoverticillium cinnamoneum: protein engineering and application for phospholipid production

This review is focusing on an industrially important enzyme, phospholipase D (PLD), exhibiting both transphosphatidylation and hydrolytic activities for various phospholipids. The transphosphatidylation activity of PLD is particularly useful for converting phosphatidylcholine (PC) into other phospholipids. During the last decade, the genes coding for PLD have been identified from various species including mammals, plants, yeast, and bacteria. However, detailed basic and applied enzymological studies on PLD have been hampered by the low productivity in these organisms. Efficient production of a recombinant PLD has also been unsuccessful so far. We recently isolated and characterized the PLD gene from Streptoverticillium cinnamoneum, producing a secretory PLD. Furthermore, we constructed an overexpression system for the secretory enzyme in an active and soluble form using Streptomyces lividans as a host for transformation of the PLD gene. The Stv. cinnamoneum PLD was proven to be useful for the continuous and efficient production of phosphatidylethanolamine (PE) from phosphatidylcholine. Thus, the secretory PLD is a promising catalyst for synthesizing new phospholipids possessing various polar head groups that show versatile physiological functions and may be utilized in food and pharmaceutical industries.     

Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.)

Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazúr (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca²⁺ ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn²⁺ ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far.     

磷脂酰丝氨酸合成酶基因pss的克隆与表达

磷脂酰丝氨酸合成酶能催化转酯反应,是定向合成特定磷脂类物质特别是磷脂酰丝氨酸的工具酶,但出发菌株产量低,很大程度上限制了酶法合成磷脂酰丝氨酸的工业化应用。利用表达载体pET-22b,实现了大肠杆菌磷脂酰丝氨酸合成酶基因在大肠杆菌BL21(DE3)中的同源高效表达。利用镍亲和柱对表达产物进行纯化,并用HPLC法对纯化后的重组酶的活力进行检测。结果表明,目的蛋白可在短时间内进行大量表达,蛋白含量是出发菌株的100倍,同时经6h的转酯反应转化率达到33%,重组磷脂酰丝氨酸合成酶活力达到69U/mg蛋白。     

Phosphatidylethanol Formation via Transphosphatidylation by Rat Brain Synaptosomal Phospholipase D

Phosphatidylethanol (Peth) formation catalyzed by the transphosphatidylation activity of phospholipase D was demonstrated to occur in a rat brain synaptosomal enriched preparation. The optimal pH was determined to be 6.5, and the optimal ethanol concentration was determined to be 0.3-0.4 M with an apparent Km of 0.2 M. Peth formation was barely detectable in the absence of an appropriate activator and several unsaturated fatty acids were found to be effective activators. The concentrations of oleic acid required for maximum activation varied with the concentration of exogenous phosphatidylcholine present in the incubation mixtures. All detergents tested were significantly less active than the unsaturated fatty acids and divalent ions were not required for Peth formation. Phosphatidylcholine was the most effective phosphatidyl donor of the phospholipids tested. Peth forming activity was greatest in the synaptic membrane fraction of the various brain subfractions examined. The 12,000 g-100,000 g particulate fraction of lung, heart, and adipose tissue had activities similar to that of brain.     

Synthesis of phosphatidylated-monoterpene alcohols catalyzed by phospholipase D and their antiproliferative effects on human cancer cells

In order to prepare functional phospholipids in the medical and pharmaceutical fields, perillyl alcohol, myrtenol, and nerol were transphosphatidylated via phospholipase D in an aqueous system. The yields of phosphatidyl-perillyl alcohol, -myrtenol, and -nerol were 79 mol %, 87 mol %, and 91 mol %, respectively. The synthetic phosphatidylated monoterpenes showed a markedly antiproliferative effect on human prostate PC-3 and human leukemia HL-60 cells at 100 μM, while the free monoterpene alcohols had no effect at 400 μM.     

Substrate specificity in phospholipid transformations by plant phospholipase D isoenzymes

Phospholipase D (PLD) catalyzes the hydrolysis and transesterification of glycerophospholipids at the terminal phosphodiester bond. In many plants, several isoforms of PLD have been identified without knowing their functional differences. In this paper, the specificities of two PLD isoenzymes from white cabbage (Brassica oleracea var. capitata) and two ones from opium poppy (Papaver somniferum L.), which were recombinantly produced in Escherichia coli, were compared in the hydrolysis of phospholipids with different head groups and in the transphosphatidylation of phosphatiylcholine with several acceptor alcohols. In a biphasic reaction system, consisting of buffer and diethyl ether, the highly homologous isoenzymes are able to hydrolyze phosphatidylcholine, -glycerol, -ethanolamine, -inositol and - with one exception - also phosphatidylserine but with different individual reaction rates. In transphosphatidylation of phosphatidylcholine, they show significant differences in the rates of head group exchange but with the same trend in the preference of acceptor alcohols (ethanolamine > glycerol ? l-serine). For l- and d-serine a stereoselectivity of PLD was observed. The results suggest a physiological relevance of the different hydrolytic and transphosphatidylation activities in plant PLD isoenzymes.     

Effect of α-tocopherol on the synthesis of phosphatidylglycerol catalyzed by phospholipase D in an aqueous system

Phosphatidylglycerol (PG) was synthesized from several phosphatidylcholines (PCs) via phospholipase D (PLD)-catalyzed transphosphatidylation in an aqueous system. The yield of PG were 71 and 68 mol% from soybean PC and egg yolk PC, respectively, under the optimum reaction conditions of 50 μmol PC, 10 mmol glycerol, 3 ml of acetate buffer, 1.6 U PLD, and 30 μmol CaCl₂ at 37°C for 48 h. In case of salmon roe PC with 14.3% eicosapentaenoic acid and 26.8% docosahexaenoic acid, the PG yield increased to 94 mol% by addition of 46 μmol α-tocopherol, although the PG yield was only 10% in absence of α-tocopherol.     

重要生化反应“转移磷脂酰反应”的研究进展

转移磷脂酰反应是在磷脂酶D的催化作用下,甘油磷脂和含羟基化合物发生碱基交换生成新的磷脂的反应。该反应为磷脂酶D所特有,被广泛的应用于动物、植物和微生物的脂类代谢、脂类信号研究以及重要生化制剂磷脂的合成工艺中。本文综述了转移磷脂酰反应的反应机制、影响因素、生物学作用及应用现状,讨论了深入研究这一反应所有待揭示的问题,并展望了今后的发展方向。     

Phospholipase D-catalyzed synthesis of new phospholipids with polar head groups

A series of new phospholipids with polar head groups have been synthesized by enzymatic transphosphatidylation of 1,2-dioleoyl-sn-glycerophosphocholine and identified by 1H NMR and MALDI-TOF-MS. The acceptor alcohols were N- or C2-substituted derivatives of ethanolamine (diethanolamine, triethanolamine, serinol, Tris, BisTris). Phospholipases D from cabbage (PLDcab) and Streptomyces sp. (PLDStr) were compared with respect to product yield and purity as well as the initial rates in transphosphatidylation and competing hydrolysis. In all reactions, PLDStr showed a remarkably higher transphosphatidylation activity than PLDcab. However, higher yields of the phospholipids with diethanolamine, triethanolamine, and serinol were obtained by PLDcab because PLDStr resulted in the additional formation of diphosphatidyl derivatives. In the synthesis of the Tris and BisTris derivatives, PLD(Str) was much more appropriate because voluminous head group alcohols (>129A3) are poorly converted by PLDcab. With BisTris as acceptor alcohol two regioisomeric forms of phosphatidyl-BisTris were obtained.     

Fatty Acid Activation and Temperature Perturbation of Rat Brain Microsomal Phospholipase D

Abstract: The hydrolytic and transphosphatidylation activities of rat brain microsomal phospholipase D were highly latent in the absence of an appropriate activator. The most suitable surfactants for this activation were oleate and palmitoleate. Besides the bile acids and unsaturated fatty acids, other naturally occurring surfactants, such as lysophospholipids, acidic phospholipids, acyl-CoA's, and gangliosides, were inactive. Taurodeoxycholate, at optimal concentration, produced a profound inhibition of oleate activation. Phospholipase D activity was detectable in all rat tissues investigated. The optimal incubation temperature for phospholipase D was 30°C, with a break point at 16.1°C in an Arrhenius plot.