Abstract
Apicomplexa are unicellular eukaryotes that are obligate, intracellular parasites of animals. Apicomplexans possess an essential lactate dehydrogenase (LDH) enzyme with a unique five amino acid insertion in the mobile, active-site specificity loop that differentiates them from homologous LDHs. This insertion contributes to the enzyme’s LDH activity, appears conserved, and is considered an attractive anti-malarial drug target. Here I investigate the insertion’s contribution to LDH activity and the functional constraints on the specificity loop. I designed and kinetically characterized an extensive collection of Plasmodium falciparum (Pf) LDH mutants. I performed an alanine scan to assess the contribution of every loop residue to LDH activity and a ConSurf analysis to rigorously analyze the putative specificity loop conservation. I designed a series of specificity loop truncation mutants to characterize the dependence of activity on loop length. Several mutants were structurally characterized by molecular replacement X-ray crystallography. Results suggest that four conserved residues, Lys102, Lys107a, Glu107e, and Trp107f, contribute to LDH activity; mutating Trp107f results in a severely crippled enzyme. The specificity loop can be truncated by as many as four residues and retain LDH activity within 100-fold of wild-type. The location of the truncation appears inconsequential and kinetic analysis indicates truncation of the specificity loop primarily affects substrate binding. The PfLDH specificity loop is extremely robust to mutation, with the exception of Trp107f. These data suggest PfLDH would likely develop resistance to drugs that do not specifically target Trp107f.