NAA50

NAA50 is a ribosome-associated N-terminal acetyltransferase (NatE catalytic subunit) that co-translationally acetylates the α-amino group of initiator methionine-retaining protein N-termini, with preference for Met-Leu, Met-Met, and other Met-Xaa sequences, and competes kinetically with methionine aminopeptidases to determine initiator methionine retention (PMID:25886145, PMID:27484799). Its GNAT-fold active site contains a methionine-specific pocket anchored by conserved histidine and tyrosine catalytic residues, and it physically associates with both subunits of the NatA complex (Naa10/Naa15) through evolutionarily conserved contacts that promote catalytic crosstalk (PMID:21900231, PMID:31155310). In addition to possessing Nα-acetyltransferase activity, NAA50 exhibits Nε-lysine acetyltransferase and autoacetylation activities that modulate its substrate specificity in vitro (PMID:19744929). In metazoans, NAA50 is essential for sister-chromatid cohesion: it N-terminally acetylates the cohesin subunit Scc1, supports the cohesin–sororin interaction, and functions antagonistically to NatA in cohesion regulation (PMID:14653991, PMID:27422821, PMID:27996020).

1. 2003 High

   Establishing that Naa50/San has a cellular function beyond generic acetyltransferase activity, Drosophila genetics revealed it is required for centromeric sister-chromatid cohesion and cohesin subunit Scc1 accumulation, and that it physically associates with the NatA complex.

   Evidence Genetic mutant analysis of san alleles in Drosophila with immunofluorescence and co-immunoprecipitation

   PMID:14653991

   Open questions at the time

   • Whether San/Naa50 acts on cohesion through its acetyltransferase activity or a non-catalytic mechanism was unresolved
   • Direct substrates relevant to cohesion were not identified
   • The nature of the NatA–Naa50 association (stoichiometry, structural basis) was unknown
2. 2009 High

   Biochemical reconstitution established that human NAA50 possesses dual Nα-terminal and Nε-lysine acetyltransferase activities, defined its preferred NAT substrate motif (Met-Leu-X-Pro), and showed that autoacetylation at specific lysines modulates substrate specificity.

   Evidence In vitro acetyltransferase assays with purified recombinant hNaa50p, oligopeptide substrate library, and mass spectrometry

   PMID:19744929

   Open questions at the time

   • In vivo relevance of KAT activity (e.g., histone H4 acetylation) was not demonstrated
   • Whether autoacetylation occurs in cells and is physiologically regulatory was untested
   • Link between enzymatic activity and cohesion phenotype remained indirect
3. 2011 High

   The first crystal structure of human Naa50 in a ternary complex with substrate peptide and CoA revealed the molecular basis of α-amino methionine specificity, identified catalytic residues (His and Tyr), and explained structural divergence from lysine acetyltransferases.

   Evidence X-ray crystallography with structure-based mutagenesis and in vitro activity assays

   PMID:21900231

   Open questions at the time

   • How Naa50 engages the ribosome and NatA complex structurally was unknown
   • Substrate specificity beyond the first residue was incompletely mapped
4. 2015 High

   In vivo acetylome profiling revealed the full breadth of Naa50 substrates (various iMet-Xaa N-termini) and demonstrated a kinetic competition with methionine aminopeptidases that determines initiator methionine retention.

   Evidence SILAC-based quantitative N-terminal acetylome profiling in yeast expressing human Naa50; in vitro MetAP competition assay

   PMID:25886145

   Open questions at the time

   • Whether the MetAP–Naa50 competition is regulated in vivo under different conditions was not addressed
   • Identification of specific endogenous substrates critical for cohesion was still missing
5. 2016 High

   Three convergent studies resolved the substrate specificity, cohesion mechanism, and NatA antagonism: crystal structures confirmed Met-Met as an optimal substrate; Drosophila genetics showed Naa50 co-translationally Nt-acetylates Scc1 to promote Scc1–Smc3 interaction; and human cell depletion experiments demonstrated that NAA50 supports the cohesin–sororin interaction and that NatA acts antagonistically to NAA50 in cohesion, with co-depletion rescuing cohesion defects.

   Evidence X-ray crystallography with tetrapeptide library (PMID:27484799); Drosophila genetics and co-IP (PMID:27996020); siRNA knockdown, epistasis rescue, chromosome spreads, and in vitro NAT assays in HeLa cells (PMID:27422821)

   PMID:27422821 PMID:27484799 PMID:27996020

   Open questions at the time

   • The molecular basis of NatA–NAA50 antagonism (distinct substrate targets) was not fully elucidated
   • Whether sororin itself is a direct Nt-acetylation substrate of NAA50 was not tested
   • How cohesion acetylation by NAA50 coordinates with Eco1/ESCO acetyltransferase-mediated cohesion establishment was unclear
6. 2019 High

   The crystal structure of the yeast NatA/Naa50 ternary complex defined the conserved binding interface between Naa50 and both NatA subunits and showed that physical association promotes catalytic crosstalk in the human but not yeast enzyme.

   Evidence X-ray crystallography of yeast NatA/Naa50 complex with in vitro NAT activity comparisons across species

   PMID:31155310

   Open questions at the time

   • A structure of the human NatA–Naa50 complex was not yet available
   • How ribosome association modulates NatE activity and crosstalk was not addressed
   • Structural basis for species-specific differences in Naa50 catalytic competence was only partially explained
7. 2020 High

   Identification and co-crystallization of selective small-molecule Naa50 inhibitors provided chemical tools and confirmed the druggability of the active site.

   Evidence DNA-encoded library screening, co-crystal structures, biochemical inhibition assays, and cellular target engagement

   PMID:32550998

   Open questions at the time

   • Cellular phenotypic consequences of chemical Naa50 inhibition (e.g., cohesion defects) were not reported
   • In vivo pharmacology and selectivity across the full NAT family were not characterized
8. 2021 High

   Structural and kinetic comparison of Arabidopsis Naa50 with yeast Naa50 pinpointed determinants of catalytic competence versus inactivity, clarifying evolutionary variation in NatE function.

   Evidence X-ray crystallography of AtNaa50 with AcCoA and bisubstrate analog; enzymatic kinetics and site-directed mutagenesis

   PMID:33400917

   Open questions at the time

   • Plant-specific substrates and physiological roles of AtNaa50 were not defined
   • Structural basis for the gain of catalytic activity in metazoan vs. fungal Naa50 remains incompletely resolved

• Key open questions include the structural basis of ribosome-associated NatE complex activity in humans, the identity of NatA substrates that antagonize NAA50-dependent cohesion, and whether NAA50's KAT activity is physiologically relevant.
• No cryo-EM or crystal structure of ribosome-bound NatE exists
• The specific NatA substrate(s) whose Nt-acetylation opposes cohesion have not been identified
• In vivo relevance of NAA50 KAT and autoacetylation activities remains untested