N-myristoyltransferases (NMTs) catalyze protein myristoylation, a major and ubiquitous lipid modification. Originally thought to modify only N-terminal glycine alpha-amino groups (G-myristoylation), NMTs are now known to modify lysine epsilon-amino groups (K-myristoylation), the significance of which is uncertain. Here we exploited systematic structural proteomics analyses and a novel pipeline involving the Shigella IpaJ protease to discriminate K- and G-myristoylation with unprecedented accuracy and identify the specific features driving each modification. NMT-dependent K-myristoylation occurs post-translationally and only on lysines 1, 2, or 3 following G-myristoylation or caspase cleavage. Direct interactions between the substrate′s reactive amino group and the NMT catalytic base slow K-myristoylation catalysis. IpaJ unmasked novel K-myristoylation sites in a dozen human proteins. The unique properties of NMT-driven K-myristoylation allowed us to design potent, mechanism-based suicide NMT inhibitors. These analyses unravel the respective paths towards K-myristoylation, G-myristoylation, or NMT inhibition, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.
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