Phospholipase D

Phospholipase D (EC 3.1.4.4, lipophosphodiesterase II, lecithinase D, choline phosphatase) (PLD) is an enzyme of the phospholipase superfamily. Phospholipases occur widely, and can be found in a wide range of organisms, including bacteria, yeast, plants, animals, and viruses. Phospholipase D’s principal substrate is phosphatidylcholine, which it hydrolyzes to produce the signal molecule phosphatidic acid (PA), and soluble choline. Plants contain numerous genes that encode various PLD isoenzymes, with molecular weights ranging from 90-125 kDa. Mammalian cells encode two isoforms of phospholipase D: PLD1 and PLD2. Phospholipase D is an important player in many physiological processes, including membrane trafficking, cytoskeletal reorganization, receptor-mediated endocytosis, exocytosis, and cell migration. Through these processes, it has been further implicated in the pathophysiology of multiple diseases: in particular the progression of Parkinson’s and Alzheimer’s, as well as various cancers.In contrast, PLD2 is a 106 kDa protein that primarily localizes to the plasma membrane, residing in light membrane lipid rafts. It has high intrinsic catalytic activity, and is only weakly activated by the above molecules.Phosphatidyl cholinePhosphatidateCholinePhospholipase cleavage sites Phospholipase D (EC 3.1.4.4, lipophosphodiesterase II, lecithinase D, choline phosphatase) (PLD) is an enzyme of the phospholipase superfamily. Phospholipases occur widely, and can be found in a wide range of organisms, including bacteria, yeast, plants, animals, and viruses. Phospholipase D’s principal substrate is phosphatidylcholine, which it hydrolyzes to produce the signal molecule phosphatidic acid (PA), and soluble choline. Plants contain numerous genes that encode various PLD isoenzymes, with molecular weights ranging from 90-125 kDa. Mammalian cells encode two isoforms of phospholipase D: PLD1 and PLD2. Phospholipase D is an important player in many physiological processes, including membrane trafficking, cytoskeletal reorganization, receptor-mediated endocytosis, exocytosis, and cell migration. Through these processes, it has been further implicated in the pathophysiology of multiple diseases: in particular the progression of Parkinson’s and Alzheimer’s, as well as various cancers. PLD-type activity was first reported in 1947 by Donald J. Hanahan and I.L. Chaikoff. It was not until 1975, however, that the hydrolytic mechanism of action was elucidated in mammalian cells. Plant isoforms of PLD were first purified from cabbage and castor bean; PLDα was ultimately cloned and characterized from a variety of plants, including rice, corn, and tomato. Plant PLDs have been cloned in three isoforms: PLDα, PLDβ, and PLDγ.More than half a century of biochemical studies have implicated phospholipase D and PA activity in a wide range of physiological processes and diseases, including inflammation, diabetes, phagocytosis, neuronal & cardiac signaling, and oncogenesis. Strictly speaking, phospholipase D is a transphosphatidylase: it mediates the exchange of polar headgroups covalently attached to membrane-bound lipids. Utilizing water as a nucleophile, this enzyme catalyzes the cleavage of the phosphodiester bond in structural phospholipids such as phosphatidylcholine and phosphatidylethanolamine. The products of this hydrolysis are the membrane-bound lipid phosphatidic acid (PA), and choline, which diffuses into the cytosol. As choline has little second messenger activity, PLD activity is mostly transduced by the production of PA. PA is heavily involved in intracellular signal transduction. In addition, some members of the PLD superfamily may employ primary alcohols such as ethanol or 1-butanol in the cleavage of the phospholipid, effectively catalyzing the exchange the polar lipid headgroup. Other members of this family are able hydrolyze other phospholipid substrates, such as cardiolipin, or even the phosphodiester bond constituting the backbone of DNA. Many of phospholipase D’s cellular functions are mediated by its principal product, phosphatidic acid (PA). PA is a negatively charged phospholipid, whose small head group promotes membrane curvature. It is thus thought to facilitate membrane-vesicle fusion and fission in a manner analogous to clathrin-mediated endocytosis. PA may also recruit proteins that contain its corresponding binding domain, a region characterized by basic amino acid-rich regions. Additionally, PA can be converted into a number of other lipids, such as lysophosphatidic acid (lyso-PA) or diacylglycerol, signal molecules which have a multitude of effects on downstream cellular pathways.PA and its lipid derivatives are implicated in myriad processes that include intracellular vesicle trafficking, endocytosis, exocytosis, actin cytoskeleton dynamics, cell proliferation differentiation, and migration. Mammalian PLD directly interacts with kinases like PKC, ERK, TYK and controls the signalling indicating that PLD is activated by these kinases. As choline is very abundant in the cell, PLD activity does not significantly affect choline levels, and choline is unlikely to play any role in signalling. Phosphatidic acid is a signal molecule and acts to recruit SK1 to membranes. PA is extremely short lived and is rapidly hydrolysed by the enzyme phosphatidate phosphatase to form diacylglycerol (DAG). DAG may also be converted to PA by DAG kinase. Although PA and DAG are interconvertible, they do not act in the same pathways. Stimuli that activate PLD do not activate enzymes downstream of DAG and vice versa. It is possible that, though PA and DAG are interconvertible, separate pools of signalling and non-signalling lipids may be maintained. Studies have suggested that DAG signalling is mediated by polyunsaturated DAG while PLD derived PA is monounsaturated or saturated. Thus functional saturated/monounsaturated PA can be degraded by hydrolysing it to form non-functional saturated/monounsaturated DAG while functional polyunsaturated DAG can be degraded by converting it into non-functional polyunsaturated PA. A lysophospholipase D called autotaxin was recently identified as having an important role in cell-proliferation through its product,lysophosphatidic acid (LPA). Plant and animal PLDs have a consistent molecular structure, characterized by sites of catalysis surrounded by an assortment of regulatory sequences. The active site of PLDs consists of four highly conserved amino acid sequences (I-IV), of which motifs II and IV are particularly conserved. These structural domains contain the distinguishing catalytic sequence HxxxxxxxKxD (HKD), where H, K, and D are the amino acids histidine (H), lysine (K), aspartic acid (D), while x represents nonconservative amino acids. These two HKD motifs confer hydrolytic activity to PLD, and are critical for its enzymatic activity both in vitro and in vivo. Hydrolysis of the phosphodiester bond occurs when these HKD sequences are in the correct proximity.