Comparative analysis of active site loop and cofactor binding site of Plasmodium vivax lactate dehydrogenase enzyme with other apicomplexan lactate dehydrogenase enzymes

Malaria is a parasitic disease that threatens almost half of the global population. It is caused by the members of the Plasmodium genus in the Apicomplexa phylum that also includes some other pathogens, Toxoplasma, Eimeria, Theileria, Cryptosporidium and Babesia.Increasing occurrence of drug resistance against malaria in many endemic areas emphasizes the need for the development of new antimalarial drugs. Mining of the metabolic pathways of the Plasmodium pointed the enzyme lactate dehydrogenase used in the glycolytic pathway of the parasite. Lactate dehydrogenase gene has been cloned from two different species of Plasmodium, the protein overexpressed and the enzyme?s structure solved in our previous studies. Structural and kinetic evaluation of the enzyme indicated that it has strikingly different properties compared to its human counterpart, and inhibition of the activity of the enzyme has been reported to kill the parasite in the erythrocyte. In this study, the active site loop that has been identified as a potential target for the structure based drug design studies and the cofactor binding site of the enzyme were analysed in Plasmodium vivax lactate dehydrogenase by mimicking the enzyme?s same regions from Toxoplasma gondii, Eimeria tenella and Theileria parva.All of the amino acid exchanges were made by site-directed mutagenesis. Cloning of the mutant genes into the expression vector pKK223-3 were performed by standart methods. All of the mutant proteins were expressed in E. coli and the expression of the proteins were confirmed by Western blotting. The overproduced mutant proteins were then purified by using Ni-NTA agarose and Steady-State kinetic analysis was performed on each mutant protein and compared with the analysis results of the wild type Plasmodium vivax lactate dehydrogenase enzyme.It was observed that making residue exchanges in the active site loop of the Plasmodium vivax lacate dehydrogenase was possible without losing enzymatic activity but enzyme?s affinity to its substrate was decreased and catalysis rate was reduced whenthe same region from Toxoplasma gondii and Eimeria tenella lactate dehydrogenases were mimicked. Making amino acid exchange on Plasmodium vivax lactate dehydrogenase to mimick Theileria parva lactate dehydrogenase has caused almost no change on the affinity of the enzyme to its substrate but improved the catalytic activity rate. These results indicate the active site loop being a crucial and ideal region across the Apicomplexan lactate dehydrogenases in the development of new antiparasitic drugs. In the second part of the study, the cofactor binding site of Plasmodium vivax lactate dehydrogenase was compared to its equivalent in the other Apicomplexan lactate dehydrogenases by replacing leucine residue at position 163 by a methionin amino acid as in the other Apicomplexan enzymes. Although the enzymatic activity was maintained after making Leucine163Methionin exchange, substrate affinity and catalytic rate of the enzyme were decreased dramatically. The enzyme has lost its activity when the leucine residue was replaced by a serine at the same position.All these results indicate active site loop as well as the cofactor binding site of the lactate dehydrogenase of Apicomplexans could be an ideal target for the structure based drug design studies.