Crystal structure of the anti-fungal target N-myristoyl transferase

Abstract

N-myristoyl transferase (NMT) catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins, and is found only in eukaryotic cells. The enzyme in this study is the 451 amino acid protein produced by Candida albicans, a yeast responsible for the majority of systemic infections in immuno-compromised humans. NMT activity is essential for vegetative growth, and the structure was determined in order to assist in the discovery of a selective inhibitor of NMT which could be developed as an anti-fungal drug. NMT has no sequence homology with other protein sequences and has a novel alpha/beta fold which shows internal two-fold symmetry, which may be a result of gene duplication. On one face of the protein there is a long, curved, relatively uncharged groove, at the center of which is a deep pocket. The pocket floor is negatively charged due to the vicinity of the C-terminal carboxylate and a nearby conserved glutamic acid residue, which separates the pocket from a cavity. These observations, considered alongside the positions of residues whose mutation affects substrate binding and activity, suggest that the groove and pocket are the sites of substrate binding and the floor of the pocket is the catalytic center.

Protein myristoylation in health and disease

“…The protein appears to have internal twofold symmetry, with two distinct but structurally similar regions corresponding to the N-and Cterminal halves. The "NMT fold" was novel at the time of its discovery [38]. A crystal structure of S. cerevisiae NMT (ScNMT) with substrate analogues of myristoyl-CoA (myrCoA) and peptides bound confirmed these observations [39].…”

“…This mechanism is supported by both biochemical and structural studies, including site-directed mutagenesis experiments [35][36][37]. An initial crystal structure was published for C. albicans NMT (CaNMT) and defined the enzyme as monomeric, with a compact globular structure and a large, saddle-shaped β-sheet flanked by several α-helices dominating the core [38]. The protein appears to have internal twofold symmetry, with two distinct but structurally similar regions corresponding to the N-and Cterminal halves.…”

Myristoyl-CoA:Protein N-Myristoyltransferase, an Essential Enzyme and Potential Drug Target in Kinetoplastid Parasites

“…The L. major and T. brucei NMTs share high similarity with other characterized NMTs in their primary sequence, including conservation of key amino acids essential for catalysis, including the "pocket floor" residues identified at the active site (10) and the hydrophobic residues (Phe 170 and Leu 171 in S. cerevisiae) critical for myristoyl-CoA binding and product release (11). The noncatalytic N termini of the parasite enzymes show sequence divergence and do not contain the polylysine domains implicated in ribosomal targeting, regions that are also missing from the NMTs of other lower eukaryotic species studied to date.…”

The Biology and Enzymology of ProteinN-Myristoylation

“…The x-ray structure of C. albicans apo-Nmt has been determined to 2.45-Å resolution (48), as have the structures of an S. cerevisiae Nmt1p⅐myristoyl-CoA binary complex (2.2 Å (45)) and two ternary complexes: Nmt1p⅐S-(2-oxo)pentadecylCoA⅐SC-58272 (2.9 Å (49)) and Nmt1p⅐S-(2-oxo)pentadecylCoA⅐GLYASKLA (2.5 Å (45)). S-(2-oxo)pentadecyl-CoA is a nonhydrolyzable myristoyl-CoA analog with a methylene group interposed between the reactive thioester carbonyl and sulfur (30).…”