Mitochondrial processing peptidases are heterodimeric enzymes (α/βMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an α/β heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single βMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas αMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric α/βMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.
In classic model organisms, cleavage of signals that are required to deliver nuclear-encoded proteins to mitochondria is mediated by an enzyme comprising two different subunits, called α or β, neither of which is functional by itself. Here, we have characterized a novel enzyme that functions in the mitosome, a highly reduced mitochondrion, of the pathogenic protist Giardia intestinalis. The Giardia enzyme is unique among eukaryotes because it has undergone reductive evolution to function efficiently as a single β-subunit monomer. We also show that the recent claim that the equivalent enzyme in the hydrogenosome, another type of reduced mitochondrion of the human parasite Trichomonas vaginalis, functions as a homodimer of two β-subunits, is not supported. The Trichomonas enzyme requires both an α- and a β-subunit to function most efficiently. Computational analysis of the Giardia and Trichomonas enzymes reveals that their structures and surface charge distributions have co-evolved to match the peculiar properties of the targeting signals that they process. The Giardia mitosome is an ideal model for studying the limits of mitochondrial reductive evolution and, because it makes cofactors that are essential for Giardia survival, is a potential therapeutic target for this important human parasite.