Cytidine Diphosphate Diacylglycerol
1. Introduction - Structure and Composition
Nucleotides are the basic building blocks of DNA and RNA, and they are required for many aspects of intermediary metabolism. They consist of three elements – a heterocyclic nitrogenous base derived from a purine (adenine and guanine) or pyrimidine (uracil, thymine and cytosine), a pentose (ribose or deoxyribose) and phosphoric acid. Cytidine, for example, consists of cytosine attached to a ribofuranose ring via a β‑N1‑glycosidic bond. From the standpoint of lipid metabolism, one of the most important of the nucleotide metabolites is cytidine 5’-phosphoric acid, which is a key component of the phospholipid cytidine diphosphate diacylglycerol (CDP-diacylglycerol or CDP-DAG), a liponucleotide. This lipid was first found in eukaryotic organisms, but it is now known to be ubiquitous and has an essential role in the biochemistry of prokaryotes also as an intermediate that occupies a branch point in the biosynthesis of certain complex glycerolipids. Its discovery owes much to serendipity allied to the receptive mind of the American biochemist Eugene Kennedy (see our web page on phosphatidylcholine).
Cytidine diphosphate diacylglycerol per se is hardly ever noticed in analyses of lipid compositions of tissues, as it is present is such small amounts, perhaps only 0.05% or so of the total phospholipids, and as a consequence, the composition of the fatty acids or molecular species in this lipid in nature (as opposed to experiments in vitro) is rarely reported. Data for ox brain are listed in Table 1. In this instance, the composition is closer to that of phosphatidylinositol, for which it is a biosynthetic precursor, than to that of any other lipid.
Table 1. Fatty acid compositions of positions sn-1 and sn-2 of the cytidine diphosphate diacylglycerol of bovine brain.
|From - Thompson, W. and MacDonald, G. Eur. J. Biochem., 65, 107-111 (1976); DOI.|
Biosynthesis of CDP-diacylglycerol (CDP-DAG) involves condensation of phosphatidic acid (PA) and cytidine triphosphate, with elimination of pyrophosphate, catalysed by an enzyme CDP-diacylglycerol synthase (CDS) (alternatively termed phosphatidate cytidylyltransferase). The other product of the reaction is pyrophosphate, which is rapidly hydrolysed irreversibly to inorganic phosphate in cells, so the reaction proceeds in one direction.
CDS is a relatively hydrophobic membrane protein that contains six transmembrane domains in eukaryotes; it is believed to function as a dimer. Two different isoforms of the enzyme associated with the outer surface of the endoplasmic reticulum and with differing tissue distributions have been found in animals, and these share approximately 71% amino acid sequence identity in humans. Of these, CDS2 is expressed ubiquitously and is selective for the acyl chains at the sn-1 and sn-2 positions and for 1-stearoyl-2-arachidonoyl-sn-phosphatidic acid in particular; this is a distinct pool utilized for phosphatidylinositide synthesis, and CDS2 is of special importance for the phosphatidylinositol cycle. Also, this isoform is particularly active in developing tissues. The most potent inhibitor of CDS2 activity, but not of CDS1, is 1-stearoyl-2-arachidonoyl-phosphatidylinositol 4,5-bisphosphate. The second isoform (CDS1) has a more restricted pattern of expression and is most active in the heart. It has no special substrate specificity and may create a separate pool of CDP-diacylglycerols for other purposes. Mutant mammalian cells lacking either CDS1 or CDS2 expression produce giant or super-sized lipid droplets in culture but by different mechanisms, although excessive phosphatidic acid production may be a factor by causing fusion of smaller lipid droplets.
A single iso-enzyme is present in yeasts and bacteria, but five forms are found in plants such as Arabidopsis, i.e. CDS1, CDS2, and CDS3 in the endoplasmic reticulum, and CDS4 and CDS5 in the chloroplasts. In yeast and animals, the loss of CDS results in lethality, but in plants a single knock-out of CDS2, CDS3, CDS4 or CDS5 did not exhibit any visible phenotype. However, double mutants of the plant genes caused death at an early stage in seedling growth.
A further distinct CDP-diacylglycerol synthase (translocator assembly and maintenance protein 41 or TAM41), first characterized in yeast, is located in the inner membrane of mitochondria facing the matrix, while a peripheral mitochondrial protein (TAMM41) is now known to be the mammalian equivalent; it bears no sequence or structural relationship to the CDS enzymes. This enzyme uses phosphatidic acid synthesised in the endoplasmic reticulum and transported into mitochondria, presumably with the aid of the contact region between the endoplasmic reticulum and mitochondria known as the mitochondria-associated membrane, and the CDP-diacylglycerol produced is used mainly for cardiolipin synthesis. Orthologues of TAM41 Have been detected in eukaryotic organisms from Drosophila to zebra fish.
The eukaryotic parasite Trypanosoma brucei has a single CDS gene and CDP-DAG is the main intermediate in the synthesis of most of its phospholipids. In the protozoan parasite, Toxoplasma gondii, there are two CDP-diacylglycerol synthases of which one is a eukaryotic type in the endoplasmic reticulum (TgCDS1) and the second a prokaryotic type (TgCDS2) in the apicoplast, a non-photosynthetic plastid-like organelle. Two discrete CDP-DAG pools are produced for the subsequent synthesis of phosphatidylinositol from the first in the Golgi bodies and phosphatidylglycerol from the second in the mitochondria. The CDS from the hyperthermophilic bacterium Thermotoga maritima was crystallized as a dimer for structural characterization, and each subunit was found to have a deep funnel-shaped cavity containing a catalytically important Mg2+,K+-hetero-di-metal center, which was open both to the cytoplasmic side of the bilayer and to the membrane. Presumably, the water-soluble and lipidic substrates (CTP, PA) and products (pyrophosphate, CDP-DAG) can enter and exit this cavity, and phosphatidic acid could potentially enter this from either the inner (cytoplasmic) or outer leaflet of the membrane. The CDP-DAG product of the reaction probably exits on the cytoplasmic side.
3. Function as a Biosynthetic Intermediate
CDP-DAG is introduced into different biosynthetic pathways to form distinct phospholipid end-products depending on the subcellular location of synthesis. In the endoplasmic reticulum, CDP-DAG is utilized immediately for the synthesis of phosphatidylinositol (PI) and of phosphatidylglycerol (PG), while in mitochondria it is used for cardiolipin synthesis via the intermediate phosphatidylglycerol. As both phosphatidylinositol and cardiolipin have highly specialized and vital roles in cells, an adequate supply of CDP-DAG is essential. Turnover is very rapid and the pool of CDP-DAG is always much smaller than that of the precursor phosphatidic acid. In contrast in animals, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triacylglycerols (TAG) are synthesised via the so-called Kennedy pathway mainly with diacylglycerols as the key intermediate, and this is also true for monogalactosyldiacylglycerols (MGDG) in plants (see the web pages on these lipids).
In fungi and prokaryotes, CDP-diacylglycerol is also the precursor for phosphatidylserine, but not in animals where there is an alternative mechanism. In yeast such as Saccharomyces cerevisiae, CDP-diacylglycerol is one of the precursors for phosphatidylethanolamine, which can in turn be converted via mono- and dimethylphosphatidylethanolamines (PME and PDME) to phosphatidylcholine, although the Kennedy pathway also functions. In the bacterium, Escherichia coli, CDP-diacylglycerols with both ribose and deoxyribose as the sugar component are produced, and both are utilized as substrates by phosphatidylserine and phosphatidylglycerophosphate synthases (no synthesis occurs via the Kennedy pathway with diacylglycerol intermediates).
It is not known whether the final fatty acid composition of the lipid is a result of the specificity of the CDP-diacylglycerol synthase in selecting particular molecular species of phosphatidic acid, or whether remodelling occurs via deacylation/re-acylation reactions as in the Lands' cycle prior to synthesis of other lipids, apart from the action of CDS2 in the biosynthesis of phosphoinositides.
Most studies of CDP-diacylglycerol have been concerned with its function as an intermediate in the biosynthesis of other lipids, and as such this is the first step in a pathway that leads only indirectly to phosphatidylethanolamine and phosphatidylcholine. In the main biosynthetic pathways to these lipids, nucleotides (CDP-ethanolamine and CDP-choline) are required but not liponucleotide intermediates. Similarly, another nucleotide, i.e. uridine 5-diphosphate(UDP)-hexose (where hexose = glucose, galactose, etc), is required for the formation of glycolipids, including both the glycosyldiacylglycerols and sphingoglycolipids.
In plants, the CDS enzymes are required for biosynthesis of phosphatidylglycerol, which is essential for photosynthesis by contributing to the stability of chlorophyll-protein complexes in thylakoid membranes.
Other functions: The extent of the biological functions of CDP-diacylglycerol, other than as an intermediate in phospholipid biosynthesis, is only now being recognized. However, CDP-DAG synthase, as a regulator of phospholipid metabolism and the rate-limiting enzyme in phosphatidylinositol biosynthesis, has a key role in the regulation of signal transduction processes dependent upon this lipid and thence indirectly in diseases related to faulty lipid metabolism. Its synthesis also influences the availability of phosphatidic acid for lipid biosynthesis, and as an example, CDP-DAG synthases have a function in controlling the size of lipid droplets in adipocytes, presumably by regulating the concentration of phosphatidic acid. In addition, CDP-DAG synthases down-regulate signalling mediated by phosphatidic acid with effects upon cell survival, proliferation, and protein synthesis. Reduced CDS1 gene expression may promote cancer.
Because it is such a minor component of tissues, isolation of cytidine diphosphate diacylglycerol appears to be a tedious task, and the only detailed published analysis of which I was aware until recently involved ion-exchange column chromatography followed by thin-layer chromatography. The pyrophosphate bond is relatively labile and is very susceptible to hydrolysis, especially under basic conditions. At natural tissue concentrations of this lipid, analysis is a challenge even with modern mass spectrometric methods, but it is possible, as has been demonstrated for the lipids of two bacterial species (cited below) .
- Blunsom, N.J. and Cockcroft, S. CDP-diacylglycerol synthases (CDS): gateway to phosphatidylinositol and cardiolipin synthesis. Front. Cell Dev. Biol., 8, 63 (2020); DOI.
- Blunsom, N.J., Gomez-Espinosa, E., Ashlin, T.G. and Cockcroft, S. Mitochondrial CDP-diacylglycerol synthase activity is due to the peripheral protein, TAMM41 and not due to the integral membrane protein, CDP-diacylglycerol synthase. Biochim. Biophys. Acta, Lipids, 1863, 284-298 (2018); DOI.
- Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Woodhead Publishing and now Elsevier) (2010) - see Science Direct.
- D'Souza, K., Kim, Y.J., Balla, T. and Epand, R.M. Distinct properties of the two isoforms of CDP-diacylglycerol synthase. Biochemistry, 53, 7358-7367 (2014); DOI.
- Jennings, W. and Epand, R.M. CDP-diacylglycerol, a critical intermediate in lipid metabolism. Chem. Phys. Lipids, 230, 104914 (2020); DOI.
- Kong, P.F., Ufermann, C.M., Zimmermann, D.L.M., Yin, Q., Suo, X., Helms, J.B., Brouwers, J.F. and Gupta, N. Two phylogenetically and compartmentally distinct CDP-diacylglycerol synthases cooperate for lipid biogenesis in Toxoplasma gondii. J. Biol. Chem., 292, 7145-7159 (2017); DOI.
- Liu, X., Yin, Y., Wu, J. and Liu, Z. Structure and mechanism of an intramembrane liponucleotide synthetase central for phospholipid biosynthesis. Nat. Commun., 5, 4244 (2014); DOI.
- Qi, Y.F., Kapterian, T.S., Du, X.M., Ma, Q.L., Fei, W.H., Zhang, Y.X., Huang, X., Dawes, I.W. and Yang, H.Y. CDP-diacylglycerol synthases regulate the growth of lipid droplets and adipocyte development. J. Lipid Res., 57, 767-780 (2016); DOI.
- Ridgway, N.D. and McLeod, R.S. (Editors) Biochemistry of Lipids, Lipoproteins and Membranes, 6th Edition. (Elsevier, Amsterdam) (2016) - several chapters - see Science Direct.
- Wang, H.Y.J., Tatituri, R.V.V., Goldner, N.K., Dantas, G. and Hsu, F.F. Unveiling the biodiversity of lipid species in Corynebacteria - characterization of the uncommon lipid families in C. glutamicum and pathogen C. striatum by mass spectrometry. Biochimie, 178, 158-169 (2020); DOI.
|Credits/disclaimer||Updated: August 11th, 2021||Author: William W. Christie|