P lasmodium falciparum SERA 5 plays a non‐enzymatic role in the malarial asexual blood‐stage lifecycle

Summary The malaria parasite P lasmodium falciparum replicates in an intraerythrocytic parasitophorous vacuole ( PV ). The most abundant P . falciparum   PV protein, called SERA 5, is essential in blood stages and possesses a papain‐like domain, prompting speculation that it functions as a proteolytic enzyme. Unusually however, SERA 5 possesses a S er residue ( S er596) at the position of the canonical catalytic C ys of papain‐like proteases, and the function of SERA 5 or whether it performs an enzymatic role is unknown. In this study, we failed to detect proteolytic activity associated with the S er596‐containing parasite‐derived or recombinant protein. However, substitution of S er596 with a C ys residue produced an active recombinant enzyme with characteristics of a cysteine protease, demonstrating that SERA 5 can bind peptides. Using targeted homologous recombination in P . falciparum , we substituted S er596 with A la with no phenotypic consequences, proving that SERA 5 does not perform an essential enzymatic role in the parasite. We could also replace an internal segment of SERA 5 with an affinity‐purification tag. In contrast, using almost identical targeting constructs, we could not truncate or C ‐terminally tag the SERA5 gene, or replace S er596 with a bulky A rg residue. Our findings show that SERA 5 plays an indispensable but non‐enzymatic role in the P . falciparum blood‐stage life cycle.