N-myristoyltransferase from Leishmania donovani: structural and functional characterisation of a potential drug target for visceral leishmaniasis.
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. donovani NMT 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 A resolution. The structure has as its defining feature a 14-stranded twisted beta-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.
Brannigan
James A
JA
York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, UK.
Smith
Barbara A
BA
Yu
Zhiyong
Z
Brzozowski
Andrzej M
AM
Hodgkinson
Michael R
MR
Maroof
Asher
A
Price
Helen P
HP
Meier
Franziska
F
Leatherbarrow
Robin J
RJ
Tate
Edward W
EW
Smith
Deborah F
DF
Wilkinson
Anthony J
AJ
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D000981:Antiprotozoal Agents / Q000494:pharmacology; D001483:Base Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D015195:Drug Design; D017125:Genes, Protozoan; D006801:Humans; D007893:Leishmania donovani / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D007898:Leishmaniasis, Visceral / Q000188:drug therapy / Q000469:parasitology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity
D000214:Acyl Coenzyme A; D000981:Antiprotozoal Agents; D017931:DNA Primers; D011994:Recombinant Proteins; D000217:Acyltransferases; C050480:glycylpeptide N-tetradecanoyltransferase