Cardiolipin, a glycerophospholipid with unique dimeric structure, is predominantly found in the mitochondrial membranes of eukaryotes and the membranes of bacteria. Cardiolipin interacts with protein complexes and plays pivotal roles in cellular energy metabolism, membrane dynamics, and stress responses. Recently, we identified the mitochondrial phosphatase, PTPMT1, as the enzyme responsible for catalyzing the conversion of phosphatidylglycerol-phosphate (PGP) to phosphatidylglycerol (PG), the penultimate step in the de novo biosynthesis of cardiolipin. Using phylogenomic analysis, we examined the evolutionary conservation of PTPMT1 and other cardiolipin biosynthetic enzymes. Our search identified a PTPMT1-like phosphatase in the bacterium Rhodopirellula baltica that could dephosphorylate PGP in vitro. Its expression restored cardiolipin deficiency and reversed growth impairment in Saccharomyces cerevisiae mutants lacking the yeast PGP phosphatase. When ectopically expressed, the bacterial PGP phosphatase localized to the mitochondria of yeast and mammalian cells. This suggests that the N-terminus of the R. baltica ortholog can be used as a mitochondrial targeting sequence in eukaryotes. Together, our study demonstrates the conservation of function between bacterial and mammalian PTPMT1 orthologs and establishes an approach to investigate the function of other cardiolipin enzymes in parasitic organism
