An unusual phosphatidylethanolamine-utilizing cardiolipin synthase is discovered in bacteria.

C ardiolipin (also known as diphosphatidylglycerol) is a four-chained anionic membrane phospholipid composed of two phosphatidyl moieties joined by a glycerol link (Fig. 1). It was first isolated from beef heart as a serologically active phospholipid (1), hence the name cardiolipin. In animals, plants, and lower eukaryotic organisms (e.g., yeast), cardiolipin is a component of the inner mitochondrial membrane bilayer, where it plays an important role in the function of enzyme complexes involved in energy transduction and ATP synthesis (2, 3). It is found in the membranes of Gram-positive and Gram-negative bacteria, where it also functions in energy production (2, 3). The nature (i.e., chain length and degree of saturation) of the four acyl groups and the hydrophilic head group of this phospholipid have an impact on mitochondrial membrane structure and function, and, by extension, the roles this organelle plays in cell physiology (2, 4). Thus, how cardiolipin is synthesized, remodeled, and metabolized are important areas of investigation. The importance of cardiolipin in mitochondrial function is further emphasized by the fact that defects in tafazzin-mediated remodeling of the fatty acyl composition of cardiolipin is the molecular basis for human Barth syndrome, a disease characterized by heart failure, myopathy, neutropenia, and growth retardation in children (3, 4). In all organisms, cardiolipin is synthesized from phosphatidylglycerol, a two-chained anionic phospholipid derived from the liponucleotide CDP-diacylglycerol (Fig. 1). How phosphatidylglycerol is converted to cardiolipin differs depending on whether cells are prokaryotic or eukaryotic. In prokaryotic cells, the cardiolipin synthase reaction involves the transfer of the phosphatidyl moiety of phosphatidylglycerol to a second molecule of phosphatidylglycerol (5), whereas, in eukaryotic cells, CDP-diacylglycerol donates its phosphatidyl moiety to phosphatidylglycerol to form cardiolipin (6, 7) (Fig. 1). In PNAS, Tan et al. (8) use a combination of genetic and biochemical approaches to discover an unusual cardiolipin synthase in the bacterium Escherichia coli by which phosphatidylethanolamine, a zwitterionic membrane phospholipid, provides the phosphatidyl group to phosphatidylglycerol to form cardiolipin (Fig. 1). Two genes, clsA (9, 10) and clsB (11), encode phosphatidylglycerol-dependent cardiolipin synthases (ClsA and ClsB, respectively) in E. coli. Although cardiolipin is undetectable in exponential phase ΔclsA ΔclsB double mutant cells, the formation of the lipid is apparent in stationary phase cells indicating a growth phase-dependent formation of cardiolipin by the newly discovered clsC gene product ClsC. This gene–enzyme relationship was identified by informatics, and confirmation was obtained through biochemical experiments that included mutagenesis of the enzyme active site (e.g., HKD phospholipase D motifs characteristic of prokaryotic cardiolipin synthases). Maximum expression of clsC-encoded activity was also dependent on the product of its neighboring gene, ymdB, but only when both are transcribed from the same operon. The mechanism by which ymdB expression stimulates ClsC activity is unclear, but is thus far unique among cardiolipin synthases. To identify the phosphatidylethanolamine-dependent nature of this unusual enzyme, Tan et al. (8) carry out a series of elegant biochemical experiments by using synthetic phospholipids with unique fatty acyl moieties and extracts of cells expressing plasmid-born clsC-ymdB but lacking the ability to synthesize phosphatidylglycerol (e.g., a ΔpgsA mutation) and cardiolipin (e.g., ΔclsA ΔclsB ΔclsC Fig. 1. Pathways for the synthesis of cardiolipin in E. coli and in yeast. In all organisms, cardiolipin is synthesized by pathways emanating from the liponucleotide CDP-diacylglycerol. In prokaryotic cells (e.g., the bacterium E. coli, Left), the final step in cardiolipin synthesis terminates with the joining of two phosphatidylglycerol molecules, but in eukaryotic cells (e.g., yeast, Right), cardiolipin is made from phosphatidylglycerol and a second molecule of CDP-diacylglycerol. In the PNAS article by Tan et al. (8), cardiolipin in E. coli is also shown to be synthesized from phosphatidylglycerol and phosphatidylethanolamine by an unusual cardiolipin synthase encoded by the clsC gene (highlighted in gray). The diagram shows the names of the genes encoding enzymes catalyzing each step in the phospholipid synthesis pathways. The structure of cardiolipin is shown with the synthetic fatty acyl groups that were used to identify the substrates of the clsC-encoded cardiolipin synthase. CL, cardiolipin; CDP-DAG, CDP-diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PGP, phosphatidylglycerophosphate; PI, phosphatidylinositol; PS, phosphatidylserine.