Abstract The homologous enzymes lactate and malate dehydrogenase (L/MDH) are structurally similar but are specific for different substrates. LDH vs MDH specificity is canonically governed by the identity of a single “specificity residue” at position 102. However, LDH function has convergently evolved from a specific MDH at least four times, and the catalytic role of residue 102 is not conserved between different phyla. The apicomplexa are a phylum of obligate, intracellular eukaryotic parasites responsible for wide-spread disease such as Plasmodium falciparum (malaria), Cryptosporidium parvum (cryptosporidiosis), Toxoplasma gondii (toxoplasmosis), and Eimeria maxima (eimeriosis). The apicomplexan LDH evolved via a five-residue insertion that produced a novel specificity residue, W107f. The commonly accepted mechanism of LDH specificity involves charge balance and steric occlusion, but our data shows that the general mechanism of apicomplexan LDHs does not use W107f as a steric block. Only Plasmodium LDHs evolved substantial steric specificity, making them exceptional among Apicomplexa. Strong protein epistasis constrained this evolution, making it difficult to revert to ancestral phenotypes. Here, we use ancestral sequence reconstruction (ASR), steady-state kinetics, and x-ray crystallography to characterize apicomplexan LDHs which challenge current assumptions about the evolution of L/MDH activity. We demonstrate the unique specificity of Plasmodium LDHs and identify the active site residues controlling their substrate recognition. The extraordinarily high specificity of Plasmodium LDHs presents difficulties for small-molecule inhibitor development, and successful drugs against Plasmodium LDH may not be efficacious against other Apicomplexa LDHs and their diseases.

Strong epistasis constrains the evolution of strict substrate specificity in apicomplexan lactate dehydrogenases