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Title: Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

Author
item PRADHAN, ANUPAM - University Of Mississippi
item TRIPATHI, ABHAI - University Of Mississippi
item DESAI, PRASHANT - University Of Mississippi
item MUKHERJEE, PRESENJIT - University Of Mississippi
item AVERY, MITCHELL - University Of Mississippi
item WALKER, LARRY - University Of Mississippi
item TEKWANI, BABU - University Of Mississippi

Submitted to: Biochimie
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/11/2009
Publication Date: 9/20/2009
Citation: Pradhan, A., Tripathi, A.K., Desai, P.V., Mukherjee, P.K., Avery, M.A., Walker, L.A., Tekwani, B.L. 2009. Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket. Biochimie. 91:1509-1517.

Interpretive Summary: The studies provide critical insights into the co-substrate binding pocket of Malate dehydrogenase from Plasmodium falciparum, the causative agent for malaria in humans. The results may be important in design of selective inhibitors of this parasite enzyme as potential antimalarial drugs.

Technical Abstract: Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal glycine motif, which forms characteristic Rossman dinucleotide-binding fold in the co-substrate binding pocket, differentiates PfMDH (GlyXGlyXXGly) from other eukaryotic and prokaryotic malate dehydrogenases (GlyXXGlyXXGly). The amino acids lining co-substrate binding pocket are completely conserved in MDHs from different species of human, primate and rodent malaria parasites. Based on this knowledge and conserved domains among prokaryotic and eukaryotic MDH, the role of critical amino acids lining co-substrate binding pocket was analyzed in catalytic functions of PfMDH by site-directed mutagenesis. Insertion of Ala at 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (characteristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH) detached regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif might not be responsible for distinct affinity of PfMDH to 3-acetylpyridine-adenine dinucleotide (APAD), a synthetic analog of NAD, since Ala9 and Ala10 insertion mutants still utilized APADH. The Gln11Met mutation, which converts the signature glycine motif in PfMDH to same as in PfLDH, did not change the enzyme function. However, Gln11Gly mutant showed about 5 fold increased catalytic activity and higher susceptibility to inhibition with gossypol. Asn119 and His174 participate in binding of both co-substrate and substrate. The Asn119Gly mutant exhibited about three fold decrease in catalytic efficiency, while mutation of His174 to Asn or alanine resulted into an inactive enzyme.