Structures of Saccharomyces cerevisiae N-myristoyltransferase with bound myristoylCoA and peptide provide insights about substrate recognition and catalysis

MyristoylCOA:protein N-myristoyltransferase (Nmt) attaches myristate to the N-terminal Gly residue of proteins involved in a variety of signal transdu ction cascades, and other critical cellular functions. To gain insight abou t the structural basis of substrate recognition and catalysis, we determine d the structures of a binary complex of Saccharomyces cerevisiae Nmt1p with myristoylCoA to 2.2 Angstrom resolution and of a ternary complex of Nmt1p with a nonhydrolyzable myristoylCoA analogue [S-(2- oxo)pentadecylCoA] and an octapeptide substrate (GLYASKLA) to 2.5 Angstrom resolution. The binary complex reveals how myristoylCoA alters the conformation of the enzyme to p romote binding of both myristoylCoA and peptide and identifies the backbone amides of F170 and L171 as an oxyanion hole which polarizes the reactive t hioester carbonyl. The ternary complex structure reveals details of the enz yme's peptide binding specificity and illuminates its mechanism of acyl tra nsfer. The N-terminal Gly ammonium is positioned in close proximity to the C-terminal carboxylate of the protein, where it is poised to undergo the re quired deprotonation to an amine. In this conformation, the nucleophile is 6.3 Angstrom away from the thioester carbonyl. A catalytic mechanism is pro posed whereby, once deprotonation is initiated, the N-terminal Gly amine ca n approximate the thioester carbonyl by rotating along Psi. This motion is facilitated by a II-bond network and leads to reaction between the glycine nitrogen nucleophile and the carbonyl. Loss of CoA from the tetrahedral int ermediate may be facilitated by intramolecular II-bonding of the sulfur to the adenylamine of CoA. This affords a compact leaving group and lends a ro le for the observed bends in the CoA structure. The absolute requirement fo r Gly at the N-terminus of substrates is explained by the requirement for f lexible rotation of its amine.