Growth phenotype analysis of heme synthetic enzymes in a halophilic archaeon, Haloferax volcanii

Halophilic euryarchaea lack many of the genes necessary for the protoporphyrin-dependent heme biosynthesis pathway previously identified in animals and plants. Bioinformatic analysis suggested the presence of two heme biosynthetic processes, an Fe-coproporphyrinogen III (coproheme) decarboxylase (ChdC) pathway and an alternative heme biosynthesis (Ahb) pathway, in Haloferax volcanii . PitA is specific to the halophilic archaea and has a unique molecular structure in which the ChdC domain is joined to the antibiotics biosynthesis monooxygenase (ABM)-like domain by a histidine-rich linker sequence. The pitA gene deletion variant of H . volcanii showed a phenotype with a significant reduction of aerobic growth. Addition of a protoheme complemented the phenotype, supporting the assumption that PitA participates in the aerobic heme biosynthesis. Deletion of the ahbD gene caused a significant reduction of only anaerobic growth by denitrification or dimethylsulfoxide (DMSO) respiration, and the growth was also complemented by addition of a protoheme. The experimental results suggest that the two heme biosynthesis pathways are utilized selectively under aerobic and anaerobic conditions in H . volcanii . The molecular structure and physiological function of PitA are also discussed on the basis of the limited proteolysis and sequence analysis.