Research paper Structure and function of Plasmodium falciparum malate dehydrogenase: Role of critical amino acids in co-substrate binding pocket q Anupam Pradhan a,1 , Abhai K. Tripathi a, 2 , Prashant V. Desai b, 3 , Prasenjit K. Mukherjee b , Mitchell A. Avery a, b , Larry A. Walker a, c , Babu L. Tekwani a, c, * a National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University MS 38677, USA b Department of Medicinal Chemistry, School of Pharmacy, University of Mississippi, University MS 38677, USA c Department of Pharmacology, School of Pharmacy, University of Mississippi, University MS 38677, USA article info Article history: Received 23 July 2009 Accepted 11 September 2009 Available online 20 September 2009 Keywords: Malate dehydrogenase Lactate dehydrogenase Gossypol Plasmodium falciparum Plasmodium spp Malaria parasite abstract The malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our laboratory 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 a 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 the 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 the co- substrate binding pocket was analyzed in catalytic functions of PfMDH using site-directed mutagenesis. Insertion of Ala at the 9th or 10th position, which converts the N-terminal GlyXGlyXXGly motif (charac- teristic of malarial MDH and LDH) to GlyXXGlyXXGly (as in bacterial and eukaryotic MDH), uncoupled regulation of the enzyme through substrate inhibition. The dinucleotide fold GlyXGlyXXGly motif seems not to be responsible for the 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 that of PfLDH, did not change the enzyme function. However, the Gln11Gly mutant showed approximately a 5-fold increase in 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 approximately a 3-fold decrease in catalytic efficiency, while mutation of His174 to Asn or Ala resulted in an inactive enzyme. These studies provide critical insights into the co-substrate binding pocket of PfMDH, which may be important in design of selective PfMDH/PfLDH inhibitors as potential antimalarials. Ó 2009 Elsevier Masson SAS. All rights reserved. 1. Introduction L-Lactate dehydrogenase (PfLDH; EC 1.1.1.27) and L-malate dehy- drogenase (PfMDH; EC 1.1.1.37), 2-hydroxy acid oxidoreductases, are important enzymes for survival and energy generation in Plasmodium falciparum, the human malaria parasite [1–3]. The former is an important enzyme of the anaerobic glucose fermentation pathway, and the latter may regulate reducing equivalents across membranes (cytoplasm and the mitochondria) and participates in malate-aspar- tate shuttle [4,5]. It is known that P. falciparum cells have mitochon- dria but do not have a fully functional Tricarboxylic Acid (TCA) cycle to generate ATP. A recent study has demonstrated that mitochondrial electron transport in the erythrocytic stages of P. falciparum is important for regeneration of ubiquinone, which is required as the electron acceptor for dihydroorotate dehydrogenase, an essential q Grant support – CDC cooperative agreements U50/CCU 423310-01, and U01/CI 000211-01; USDA-ARS scientific cooperative agreement no 58-6408-2-0009. * Corresponding author at: National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University MS 38655. Tel.: þ1 662 915 7882; fax: þ1 662 915 7062. E-mail address: btekwani@olemiss.edu (B.L. Tekwani). 1 Present address: Department of Global Health, College of Public Health, University of South Florida, Tampa, FL 33612, USA. 2 Present address: Department of Molecular Microbiology and Immunology, John Hopkins University, Baltimore, MD 21205, USA. 3 Present address: Lilly Research Laboratories, Lilly Corporate Center, Indian- apolis, IN 46285, USA. Contents lists available at ScienceDirect Biochimie journal homepage: www.elsevier.com/locate/biochi 0300-9084/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.09.005 Biochimie 91 (2009) 1509–1517