Implications for the evolution of eukaryotic amino-terminal acetyltransferase (NAT) enzymes from the structure of an archaeal ortholog

Abstract

Amino-terminal acetylation is a ubiquitous modification in eukaryotes that is involved in a growing number of biological processes. There are six known eukaryotic amino-terminal acetyltransferases (NATs), which are differentiated from one another on the basis of substrate specificity. To date, two eukaryotic NATs, NatA and NatE, have been structurally characterized, of which NatA will acetylate the α-amino group of a number of nonmethionine amino-terminal residue substrates such as serine; NatE requires a substrate amino-terminal methionine residue for activity. Interestingly, these two NATs use different catalytic strategies to accomplish substrate-specific acetylation. In archaea, where this modification is less prevalent, only one NAT enzyme has been identified. Surprisingly, this enzyme is able to acetylate NatA and NatE substrates and is believed to represent an ancestral NAT variant from which the eukaryotic NAT machinery evolved. To gain insight into the evolution of NAT enzymes, we determined the X-ray crystal structure of an archaeal NAT from Sulfolobus solfataricus (ssNAT). Through the use of mutagenesis and kinetic analysis, we show that the active site of ssNAT represents a hybrid of the NatA and NatE active sites, and we highlight features of this protein that allow it to facilitate catalysis of distinct substrates through different catalytic strategies, which is a unique characteristic of this enzyme. Taken together, the structural and biochemical data presented here have implications for the evolution of eukaryotic NAT enzymes and the substrate specificities therein.structural biology | evolutionary biology | enzymology | X-ray crystallography T he cotranslational process of amino-terminal acetylation occurs on a majority of eukaryotic proteins and mediates many biological processes, including cellular apoptosis, enzymatic regulation, protein localization, and protein degradation (1-4). In humans, three major amino-terminal acetyltransferase (NAT) complexes, called NatA, NatB, and NatC, are responsible for modifying ∼85% of all proteins that undergo amino-terminal acetylation (5). These complexes are conserved in yeast, exist as obligate heterodimers, and are differentiated from one another on the basis of substrate specificity, which is dictated by the amino-terminal sequence of the substrate protein (5, 6). Each complex consists of a single unique catalytic subunit and an additional unique auxiliary subunit that has been shown to potentiate activity and alter substrate specificity of the enzymatic component, as well as anchor the complex to the ribosome during translation (6-11). Three additional human NAT enzymes, NatD-NatF, have also been identified, but they have a much more limited set of physiological substrates, appear to be independently active, and are not well characterized across eukaryotes (12)(13)(14).The only two eukaryotic NATs that have been structurally characterized are Schizosaccharomyces pombe NatA, consisting of the Naa10p catalytic subunit and Naa15p regulatory subunit, and ...

The biological functions of Naa10 — From amino-terminal acetylation to human disease

“…In line with this, in vitro acetylation assays showed that, although ssNaa10 has higher sequence similarity to hNaa10 over hNaa50 (34% vs. 24% sequence identity, respectively) and therefore has a preference for Ser- over Met-amino-terminal substrates, it is still able to accommodate NatA and NatE substrates (Liszczak and Marmorstein, 2013). Furthermore, studies on the X-ray crystal structure of ssNaa10 combined with mutagenesis and kinetic analyses showed that the active site of ssNaa10 represents a hybrid of the NatA and NatE active sites (Liszczak and Marmorstein, 2013), explaining the above findings. A later study confirmed these findings and showed that Glu35 has a critical role for the unique substrate specificity of ssNaa10 (Chang and Hsu, 2015).…”

“…In the archeae Sulfolobus solfataricus , only a single ssNaa10 has been identified to date with a more relaxed substrate specificity (acetylating NatA, NatB and NatC-type substrates) (Mackay et al, 2007). A recent structural comparison of ssNaa10 with S. pombe Naa10 showed that the active site of ssNaa10 represents a hybrid of Naa10 and Naa50 and, as a result, can facilitate acetylation of Met- and non-Met-containing amino-terminal substrate peptides (Liszczak and Marmorstein, 2013). Therefore, the authors suggest that this ssNaa10 is an ancestral NAT variant from which the eukaryotic NAT machinery evolved (Liszczak and Marmorstein, 2013).…”

The N-terminal Acetyltransferase Naa10/ARD1 Does Not Acetylate Lysine Residues

“…These include the transcription factor Runt-related transcription factor 2 (Runx2) (11), the enzyme methionine sulfoxide reductase A (MSRA) (12), and myosin light chain kinase (MLCK) (13). These findings were surprising to us because the structures of all NATs determined to date (9, 14 -17), including Naa10 (9), contain an extended loop that seems to occlude lysine side chains within a polypeptide from lying across the active site as they do in KATs (15)(16)(17)(18). In addition, other reports demonstrating that Naa10 acetylates a lysine residue on Hif-1␣ have failed to be replicated (19 -21), making the question of whether Naa10 is able to acetylate lysine residues controversial.…”

A Saccharomyces cerevisiae Model Reveals In Vivo Functional Impairment of the Ogden Syndrome N-Terminal Acetyltransferase NAA10 Ser37Pro Mutant

“…Recently, the first structures of NATs and a NAT-complex were solved, providing a molecular understanding of the sequence specific Nt-acetylation of protein N termini (27)(28)(29)(30). Structural analyses of noncomplexed Naa10 and NatA from Schizosaccharomyces pombe reveal an allosteric modulator function of Naa15 in steering Naa10 specificity and provide a rational for the distinctive substrate specificity profiles observed when assaying non-complexed versus complexed Naa10 (10,27), with both forms co-existing in cells (10).…”