N-Myristoyltransferase from Leishmania donovani: Structural and Functional Characterisation of a Potential Drug Target for Visceral Leishmaniasis

Abstract

N-Myristoyltransferase (NMT) catalyses the attachment of the 14-carbon saturated fatty acid, myristate, to the amino-terminal glycine residue of a subset of eukaryotic proteins that function in multiple cellular processes, including vesicular protein trafficking and signal transduction. In these pathways, N-myristoylation facilitates association of substrate proteins with membranes or the hydrophobic domains of other partner peptides. NMT function is essential for viability in all cell types tested to date, demonstrating that this enzyme has potential as a target for drug development. Here, we provide genetic evidence that NMT is likely to be essential for viability in insect stages of the pathogenic protozoan parasite, Leishmania donovani, causative agent of the tropical infectious disease, visceral leishmaniasis. The open reading frame of L. donovaniNMT has been amplified and used to overproduce active recombinant enzyme in Escherichia coli, as demonstrated by gel mobility shift assays of ligand binding and peptide-myristoylation activity in scintillation proximity assays. The purified protein has been crystallized in complex with the non-hydrolysable substrate analogue S-(2-oxo)pentadecyl-CoA, and its structure was solved by molecular replacement at 1.4 Å resolution. The structure has as its defining feature a 14-stranded twisted β-sheet on which helices are packed so as to form an extended and curved substrate-binding groove running across two protein lobes. The fatty acyl-CoA is largely buried in the N-terminal lobe, its binding leading to the loosening of a flap, which in unliganded NMT structures, occludes the protein substrate binding site in the carboxy-terminal lobe. These studies validate L. donovani NMT as a potential target for development of new therapeutic agents against visceral leishmaniasis.

Association of NMT2 with the acyl-CoA carrier ACBD6 protects the N-myristoyltransferase reaction from palmitoyl-CoA

“…Because binding of the peptides does not affect acyl-CoA binding to the N-terminal motif ( 33,53,54 ), compounds that can recognize the C-terminal binding domain of the NMT proteins of invading pathogens seem to be good candidates to specifi cally disrupt myristoylation without impacting acylation of the proteins of the host (28)(29)(30)(31)(32)(34)(35)(36). As examples, the development of Plasmodium falciparum in erythroid cells is blocked by several chemicals inhibiting the P. falciparum NMT enzyme (35)(36)(37)55 ).…”

“…Although amide linkage of the myristate to the glycine residue appears irreversible, a structural conformational property of some acyl-proteins, called the myristoylswitch, can remove the aliphatic tail from the lipid bilayer and dissociate the proteins from their membrane-bound location ( 25,27 ). The acyl-transferase activity of the NMT enzymes is specifi c toward myristoyl-CoA (C 14 -CoA) and is essential for the intracellular development of pathogens (28)(29)(30)(31)(32)(33)(34)(35)(36). Chemicals inhibiting myristoylation of proteins are potent drugs against parasitic protozoa and fungi.…”

Design of Novel N-Myristoyltransferase Inhibitors of Leishmania donovani Using Four-Dimensional Quantitative Structure-Activity Relationship Analysis

“…The crystal structural coordinates of NMT at 1.42 Å resolution was obtained from the Protein Data Bank (PDB code: 2WUU), 5 and the protein has been crystallized in complex with the non-hydrolysable substrate analogue S-(2-oxo)pentadecyl-CoA. For the docking procedure the Molegro Virtual Docker (MVD) program was used.…”

“…4 N-Myristoylation plays an important role in protein-protein interactions of membrane proteins which, in turn, facilitate a variety of signal transduction pathways. 4 NMT has been found to be essential in Leishmania donovani life cycle, 5 becoming highly promising as a drug target for the treatment of VL. 6 The design of new bioactive molecules can be accomplished by several different methods, one of which is the quantitative structure activity relationship (QSAR), which relates biological data to the chemical structure.…”

N-myristoyltransferase in the leukocytic development processes

“…The N-terminal glycine myristoylation of various key proteins is necessary for normal cell functioning, and thus NMT is essential for survival and growth in a number of organisms (Duronio et al 1989;Lodge et al 1994;Weinberg et al 1995;Boutin 1997;Wright et al 2010). In organisms in which a single NMT isoform exists, the targeting of the endogenous NMT functions has been the candidate of choice for the treatment of many human pathogenic states with a focus on developing species-specific NMT inhibitors as anti-fungal, antiparasitic, and antiviral agents (Duronio et al 1991;Sikorski et al 1997;Lodge et al 1998;Georgopapadakou 2002;Price et al 2003;Gelb et al 2003;French et al 2004;Panethymitaki et al 2006;Bowyer et al 2007;Brannigan et al 2010;Frearson et al 2010). In the higher eukaryotes, the two isoforms NMT1 and NMT2 have overlapping but distinct substrate specificities (Giang and Cravatt 1998;Ducker et al 2005).…”