Abstract
Apicomplexa are unicellular eukaryotic parasites of many animals, including humans. The most notorious example is malaria, caused by the apicomplexan parasite Plasmodium falciparum. The apicomplexan lactate dehydrogenase enzyme (LDH) evolved independently from human and other "canonical" LDHs found in the host. Apicomplexan LDHs have distinct structures from the host LDHs, and thus they have been identified as a drug target for the parasitic diseases. Plasmodium falciparum LDH (PfLDH) has been extensively studied due to its value for drug targeting, and yet the mechanistic and evolutionary basis of the functional and structural divergence of the entire apicomplexan LDH family is still not well-understood. A related apicomplexan parasite, Eimeria maxima, is the cause of an economically significant disease of poultry. To further explore the source of the functional divergence in apicomplexan LDHs, I characterized the kinetic and specificity profile for PfLDH, Eimeria maxima LDH (EmLDH), and their most recent common ancestor (AncLDH) calculated from extant sequences, using different substrates. PfLDH diverged from AncLDH by evolving high specificity toward pyruvate, having very low activities toward other bigger and hydrophobic substrates. EmLDH, on the other hand, largely retains the specificity profile from the AncLDH ancestor: both EmLDH and AncLDH have reduced activities toward other alternative substrates, but the magnitudes were no match to PfLDH. The most prominent difference can be seen when phenylpyruvate was used as an alternative substrate. PfLDH has very limited activity toward this hydrophobic substrate, while AncLDH and EmLDH are both able to ignore the phenyl ring and turn over phenylpyruvate at an efficiency only marginally lower than turning over the native substrate pyruvate. I have also solved the first crystal structure for EmLDH (1.8 Å). Structural comparison of EmLDH, PfLDH, and AncLDH shows several residue differences near the active site, and it is likely that the differences in their specificity profiles can be attributed to these residues. Preliminary data also suggests that the rate-limiting step for EmLDH is after the hydride transfer step, similar to PfLDH. This work will serve as a foundation for further understanding the origin of the convergent evolution of LDH function in the Apicomplexa.