Specific Inhibition of the Aspartate Aminotransferase of Plasmodium falciparum

• Published in: Journal of molecular biology 2011-01, Vol.405 (4), p.956-971
• Main Authors: Wrenger, Carsten, Müller, Ingrid B., Schifferdecker, Anna J., Jain, Rishabh, Jordanova, Rositsa, Groves, Matthew R.
• Format: Article
• Language: English
• Subjects: active sites; Amino Acid Sequence; Animals; Antimalarials - pharmacology; aspartate aminotransferase; Aspartate Aminotransferases - antagonists & inhibitors; Aspartate Aminotransferases - chemistry; Aspartate Aminotransferases - genetics; aspartate transaminase; aspartic acid; Base Sequence; biosynthesis; catalytic activity; Crystallography, X-Ray; cytosol; Cytosol - enzymology; Dimerization; DNA Primers - genetics; DNA, Protozoan - genetics; drugs; energy metabolism; enzyme inhibition; Enzyme Inhibitors - pharmacology; glutamic acid; Humans; in vitro inhibition; In Vitro Techniques; malaria; Malaria, Falciparum - drug therapy; Malaria, Falciparum - parasitology; malates; mitochondria; Models, Molecular; Molecular Sequence Data; nitrogen metabolism; parasites; Plasmodium falciparum; Plasmodium falciparum - drug effects; Plasmodium falciparum - enzymology; Plasmodium falciparum - genetics; Protein Conformation; Protein Interaction Domains and Motifs; Protein Structure, Quaternary; pyrimidines; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Sequence Deletion; Sequence Homology, Amino Acid; Species Specificity; Static Electricity; subcellular localization; X-ray crystallography
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2010.11.018

Aspartate aminotransferases (AspATs; EC 2.6.1.1) catalyze the conversion of aspartate and α-ketoglutarate into oxaloacetate and glutamate and are key enzymes in the nitrogen metabolism of all organisms. Recent findings suggest that the plasmodial enzyme [Plasmodium falciparum aspartate aminotransferase (PfAspAT)] may also play a pivotal role in energy metabolism and in the de novo biosynthesis of pyrimidines. However, while PfAspAT is a potential drug target, the high homology between the active sites of currently available AspAT structures hinders the development of specific inhibitors of these enzymes. In this article, we report the X-ray structure of the PfAspAT homodimer at a resolution of 2.8 Å. While the overall fold is similar to the currently available structures of other AspATs, the structure presented shows a significant divergence in the conformation of the N-terminal residues. Deletion of these divergent PfAspAT N-terminal residues results in a loss of activity for the recombinant protein, and addition of a peptide containing these 13 N-terminal residues results in inhibition both in vitro and in a lysate isolated from cultured parasites, while the activity of human cytosolic AspAT is unaffected. The finding that the divergent N-terminal amino acids of PfAspAT play a role in catalytic activity indicates that specific inhibition of the enzyme may provide a lead for the development of novel compounds in the treatment of malaria. We also report on the localization of PfAspAT to the parasite cytosol and discuss the implications of the role of PfAspAT in the supply of malate to the parasite mitochondria. [Display omitted] ► We present the 2.8-Å structure of the aspartate aminotransferase of P. falciparum (PfAspAT) as determined by X-ray crystallography. ► We have performed comparative in vivo and in vitro activity assays of PfAspAT and human cytosolic AspAT (HscAspAT). ► We demonstrate specific inhibition of PfAspAT through disruption of the N-terminal amino acids. ► We present data that localizes PfAspAT to the parasitic cytosol.