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Cardiolipins (CL) are a class of lipids involved in the structural organization of membranes, enzyme functioning, and osmoregulation. Biosynthesis of CLs has been studied in eukaryotes and bacteria, but has been barely explored in archaea. Unlike the common fatty acyl chain-based ester phospholipids, archaeal membranes are made up of the structurally different isoprenoid-based ether phospholipids, possibly involving a different cardiolipin biosynthesis mechanism. Here, we identified a phospholipase D motif-containing cardiolipin synthase (MhCls) from the methanogen Methanospirillum hungatei. The enzyme was overexpressed in Escherichia coli, purified, and its activity was characterized by LC-MS analysis of substrates/products. MhCls utilizes two archaetidylglycerol (AG) molecules in a transesterification reaction to synthesize glycerol-di-archaetidyl-cardiolipin (Gro-DACL) and glycerol. The enzyme is non-selective to the stereochemistry of the glycerol-backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol (PG) to generate glycerol-di-phosphatidyl-cardiolipin (Gro-DPCL). Remarkably, in the presence of AG and PG, MhCls formed glycerol-archaetidyl-phosphatidyl-cardiolipin (Gro-APCL), an archaeal-bacterial hybrid cardiolipin species that so far has not been observed in nature. Due to the reversibility of the transesterification, in the presence of glycerol, Gro-DPCL can be converted back into two PG molecules. In the presence of other compounds that contain primary hydroxyl groups (e.g., alcohols, water, sugars), various natural and unique unnatural phospholipid species could be synthesized, including multiple di-phosphatidyl-cardiolipin species. Moreover, MhCls can utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphatidyl-cardiolipin species, emphasizing the promiscuity of this cardiolipin synthase, that could be of interest for bio-catalytic purposes.

Cardiolipin (DPCL) biosynthesis has barely been explored in Archaeal isoprenoid-based ether lipid membranes. Here, we identified a cardiolipin synthase (MhCls) from the mesophilic anaerobic methanogenMethanospirillum hungatei.The enzyme was overexpressed inEscherichia coli,purified, and subsequently characterized by LC-MS. MhCls utilizes two archaetidylglycerol molecules in a transesterification reaction to synthesize archaeal di-phosphate cardiolipin (aDPCL) and glycerol. The enzyme is invariant to the stereochemistry of the glycerol-backbone and the nature of the lipid tail, as it also accepts phosphatidylglycerol to generate di-phosphate cardiolipin (DPCL). Remarkably, in the presence of archaetidylglycerol and phosphatidylglycerol, MhCls formed an archaeal-bacterial hybrid di-phosphate cardiolipin (hDPCL), that so far has not been observed in nature. Due to the reversibility of the transesterification, cardiolipin can be converted back in presence of glycerol into phosphatidylglycerol. In the presence of other compounds that contain primary hydroxyl groups (e.g. alcohols, water, sugars) various natural and unique artificial phospholipid species could be synthesized, including multiple di-phosphate cardiolipin species. Moreover, MhCls could utilize a glycolipid in the presence of phosphatidylglycerol to form a glycosyl-mono-phosphate cardiolipin, emphasizing the promiscuity of this cardiolipin synthase.