Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria parasites

The malaria parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of GPI anchor biosynthesis, the putative phosphomannomutase (EC 5.4.2.8) of the parasites, HAD5 (PF3D7_1017400). We confirm the phosphomannomutase and phosphoglucomutase activity of purified recombinant HAD5. By regulating expression of HAD5 in transgenic parasites, we demonstrate that HAD5 is required for malaria parasite egress and erythrocyte reinvasion. Finally, we determine the three-dimensional crystal structure of HAD5 and identify a substrate analog that specifically inhibits HAD5, compared to orthologous human phosphomannomutases. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition, and that HAD5 has potential as a multi-stage antimalarial target. ### Competing Interest Statement The authors have declared no competing interest. * aTc : anhydrotetracycline CSP : circumsporozoite protein E : ethanolamine E64 : epoxysuccinyl-L-leucylamido(4-guanidino)butane FSM : fosmidomycin G-1,6-P : glucose-1,6-bisphosphate G1P : glucose 1-phosphate G6P : glucose 6-phosphate GlcN : glucosamine GPI : glycosylphosphatidylinositol GPI-AP : GPI-anchored protein HAD : haloacid dehalogenase Ins : inositol LIC : ligation-independent cloning M1P : mannose 1-phosphate M6P : mannose 6-phosphate Man : mannose ManN : mannosamine MSP : merozoite surface protein PGM : phosphoglucomutase PGP : phosphoglycolate phosphatase PMM : phosphomannomutase RAMA : rhoptry-associated membrane antigen