NATs at a glance.

Most proteins receive an acetyl group at the N terminus while in their nascency as the result of modification by co-translationally acting N-terminal acetyltransferases (NATs). The N-terminal acetyl group can influence several aspects of protein functionality. From studies of NAT-lacking cells, it is evident that several cellular processes are affected by this modification. More recently, an increasing number of genetic cases have demonstrated that N-terminal acetylation has crucial roles in human physiology and pathology. In this Cell Science at a Glance and the accompanying poster, we provide an overview of the human NAT enzymes and their properties, substrate coverage, cellular roles and connections to human disease.

Structural insights of human N-acetyltransferase 10 and identification of its potential novel inhibitors.

N-acetyltransferase 10 (NAT10), is an acetyltransferase that regulates RNA stability and translation processes. Association of NAT10 with several diseases including cancer, makes it a promising therapeutic target. Remodelin is the only known NAT10 inhibitor, but the structural information related to its binding with NAT10 is still obscure. Here, we predicted the human NAT10 structure using homology modeling that was not available previously and used human NAT10 to identify the novel binding site(s) of Remodelin. The alignment of the modeled human NAT10 showed 24% identity and 37% positivity with crystal structure of tRNA (Met) cytidine acetyltransferase. Molecular docking showed binding of Remodelin with NAT10 in acetyl-CoA binding pocket. Additionally, we screened a library of FDA-approved drugs for the identification of novel inhibitors of NAT10 activity. Binding score showed that four drugs namely, Fosaprepitant (- 11.709), Leucal (- 10.46), Fludarabine (- 10.347) and Dantrolene (- 9.875) bind to NAT10 and have better binding capability when compared with Acetyl-CoA (- 5.691) and Remodelin (- 5.3). Acetyl-CoA, Remodelin, and others exhibit hits for hydrophobic, hydrophilic and hydrogen interactions. Interestingly, Remodelin and others interact with the amino acid residues ILE629, GLY639, GLY641, LEU719, and PHE722 in the Acetyl-CoA binding pocket of NAT10 similar to Acetyl-CoA. Our findings revealed that Fosaprepitant, Leucal, Fludarabine, and Dantrolene are promising molecules that can be tested and developed as potential inhibitors of NAT10 acetyltransferase activity.

Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation.

Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-α-acetylation (NTA) and ε-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities.

Biochemical and structural analysis of N-terminal acetyltransferases.

N-terminal acetylation is a co- and post-translational modification catalyzed by the conserved N-terminal acetyltransferase (NAT) family of enzymes. A majority of the human proteome is modified by the human NATs (NatA-F and H), which are minimally composed of a catalytic subunit and as many as two auxiliary subunits. Together, NATs specifically regulate many cellular functions by influencing protein activities such as their degradation, membrane targeting, and protein-protein interactions. This chapter will describe methods developed for their preparation, and their biochemical and structural characterization. This will include methodologies for expression and purification of recombinant NAT protein, kinetic assays, biochemical and biophysical assays, and strategies for structural studies.

Recent Advances on the Structure and Function of RNA Acetyltransferase Kre33/NAT10.

Ribosome biogenesis is one of the most energy demanding processes in the cell. In eukaryotes, the main steps of this process occur in the nucleolus and include pre-ribosomal RNA (pre-rRNA) processing, post-transcriptional modifications, and assembly of many non-ribosomal factors and ribosomal proteins in order to form mature and functional ribosomes. In yeast and humans, the nucleolar RNA acetyltransferase Kre33/NAT10 participates in different maturation events, such as acetylation and processing of 18S rRNA, and assembly of the 40S ribosomal subunit. Here, we review the structural and functional features of Kre33/NAT10 RNA acetyltransferase, and we underscore the importance of this enzyme in ribosome biogenesis, as well as in acetylation of non-ribosomal targets. We also report on the role of human NAT10 in Hutchinson-Gilford progeria syndrome.

A Gcn5-Related N-Acetyltransferase (GNAT) Capable of Acetylating Polymyxin B and Colistin Antibiotics in Vitro.

Deeper exploration of uncharacterized Gcn5-related N-acetyltransferases has the potential to expand our knowledge of the types of molecules that can be acylated by this important superfamily of enzymes and may offer new opportunities for biotechnological applications. While determining native or biologically relevant in vivo functions of uncharacterized proteins is ideal, their alternative or promiscuous in vitro capabilities provide insight into key active site interactions. Additionally, this knowledge can be exploited to selectively modify complex molecules and reduce byproducts when synthetic routes become challenging. During our exploration of uncharacterized Gcn5-related N-acetyltransferases from Pseudomonas aeruginosa, we identified such an example. We found that the PA3944 enzyme acetylates both polymyxin B and colistin on a single diaminobutyric acid residue closest to the macrocyclic ring of the antimicrobial peptide and determined the PA3944 crystal structure. This finding is important for several reasons. (1) To the best of our knowledge, this is the first report of enzymatic acylation of polymyxins and thus reveals a new type of substrate that this enzyme family can use. (2) The enzymatic acetylation offers a controlled method for antibiotic modification compared to classical promiscuous chemical methods. (3) The site of acetylation would reduce the overall positive charge of the molecule, which is important for reducing nephrotoxic effects and may be a salvage strategy for this important class of antibiotics. While the physiological substrate for this enzyme remains unknown, our structural and functional characterization of PA3944 offers insight into its unique noncanonical substrate specificity.

Actin's N-terminal acetyltransferase uncovered.

Humans express six highly conserved actin isoforms, which differ the most at their N-termini. Actin's N-terminus undergoes co- and post-translational processing unique among eukaryotic proteins. During translation, the initiator methionine of the two cytoplasmic isoforms is N-terminally acetylated (Nt-acetylated) and that of the four muscle isoforms is removed and the exposed cysteine is Nt-acetylated. Then, an unidentified acetylaminopeptidase post-translationally removes the Ac-Met (or Ac-Cys), and all six isoforms are re-acetylated at the N-terminus. Despite the vital importance of actin for cellular processes ranging from cell motility to organelle trafficking and cell division, the mechanism and functional consequences of Nt-acetylation remained unresolved. Two recent studies significantly advance our understanding of actin Nt-acetylation. Drazic et al. (2018, Proc Natl Acad Sci U S A, 115, 4399-4404) identify actin's dedicated N-terminal acetyltransferase (NAA80/NatH), and demonstrate that Nt-acetylation critically impacts actin assembly in vitro and in cells. NAA80 knockout cells display increased filopodia and lamellipodia formation and accelerated cell motility. In vitro, the absence of Nt-acetylation leads to a decrease in the rates of filament depolymerization and elongation, including formin-induced elongation. Goris et al. (2018, Proc Natl Acad Sci U S A, 115, 4405-4410] describe the structure of Drosophila NAA80 in complex with a peptide-CoA bi-substrate analog mimicking the N-terminus of ß-actin. The structure reveals the source of NAA80's specificity for actin's negatively-charged N-terminus. Nt-acetylation neutralizes a positive charge, thus enhancing the overall negative charge of actin's unique N-terminus. Actin's N-terminus is exposed in the filament and influences the interactions of many actin-binding proteins. These advances open the way to understanding the many likely consequences and functional roles of actin Nt-acetylation.

Spotlight on protein N-terminal acetylation.

N-terminal acetylation (Nt-acetylation) is a widespread protein modification among eukaryotes and prokaryotes alike. By appending an acetyl group to the N-terminal amino group, the charge, hydrophobicity, and size of the N-terminus is altered in an irreversible manner. This alteration has implications for the lifespan, folding characteristics and binding properties of the acetylated protein. The enzymatic machinery responsible for Nt-acetylation has been largely described, but significant knowledge gaps remain. In this review, we provide an overview of eukaryotic N-terminal acetyltransferases (NATs) and the impact of Nt-acetylation. We also discuss other functions of known NATs and outline methods for studying Nt-acetylation.

Structural determinants and cellular environment define processed actin as the sole substrate of the N-terminal acetyltransferase NAA80.

N-terminal (Nt) acetylation is a major protein modification catalyzed by N-terminal acetyltransferases (NATs). Methionine acidic N termini, including actin, are cotranslationally Nt acetylated by NatB in all eukaryotes, but animal actins containing acidic N termini, are additionally posttranslationally Nt acetylated by NAA80. Actin Nt acetylation was found to regulate cytoskeletal dynamics and motility, thus making NAA80 a potential target for cell migration regulation. In this work, we developed potent and selective bisubstrate inhibitors for NAA80 and determined the crystal structure of NAA80 in complex with such an inhibitor, revealing that NAA80 adopts a fold similar to other NAT enzymes but with a more open substrate binding region. Furthermore, in contrast to most other NATs, the substrate specificity of NAA80 is mainly derived through interactions between the enzyme and the acidic amino acids at positions 2 and 3 of the actin substrate and not residues 1 and 2. A yeast model revealed that ectopic expression of NAA80 in a strain lacking NatB activity partially restored Nt acetylation of NatB substrates, including yeast actin. Thus, NAA80 holds intrinsic capacity to posttranslationally Nt acetylate NatB-type substrates in vivo. In sum, the presence of a dominant cotranslational NatB in all eukaryotes, the specific posttranslational actin methionine removal in animals, and finally, the unique structural features of NAA80 leave only the processed actins as in vivo substrates of NAA80. Together, this study reveals the molecular and cellular basis of NAA80 Nt acetylation and provides a scaffold for development of inhibitors for the regulation of cytoskeletal properties.

Homology modeling and prediction of the amino acid residues participating in the transfer of acetyl-CoA to arylalkylamine by the N-acetyltransferase from Chryseobacterium sp.

OBJECTIVES: To predict the amino acid residues playing important roles in acetyl-CoA and substrate binding and to study the acetyl group transfer mechanism of Chryseobacterium sp. 5-3B N-acetyltransferase (5-3B NatA). RESULTS: A 3-dimensional homology model of 5-3B NatA was constructed to compare the theoretical structure of this compound with the structures of previously reported proteins belonging to the bacterial GCN5 N-acetyltransferase family. Homology modeling of the 5-3B NatA structure and a characterization of the enzyme's kinetic parameters identified the essential amino acid residues involved in binding and acetyl-group transfer. 126Leu, 132Leu, and 135Lys were implicated in the binding of phosphopantothenic acid, and 100Tyr and 131Lys in that of adenosyl biphosphate. The data supported the participation of 83Glu and 133Tyr in catalyzing acetyl-group transfer to L-2-phenylglycine. CONCLUSIONS: 5-3B NatA catalyzes the enantioselective N-acetylation of L-2-phenylglycine via a ternary complex comprising the enzyme, acetyl-CoA, and the substrate.

DTNB-Based Quantification of In Vitro Enzymatic N-Terminal Acetyltransferase Activity.

We here describe a quick and easy method to quantitatively measure in vitro acetylation activity of not only N-terminal acetyltransferase (NAT) enzymes, but acetyltransferases using acetyl-coenzyme A as an acetyl donor in general.

Biochemical evidence for relaxed substrate specificity of Nα-acetyltransferase (Rv3420c/rimI) of Mycobacterium tuberculosis.

Nα-acetylation is a naturally occurring irreversible modification of N-termini of proteins catalyzed by Nα-acetyltransferases (NATs). Although present in all three domains of life, it is little understood in bacteria. The functional grouping of NATs into six types NatA - NatF, in eukaryotes is based on subunit requirements and stringent substrate specificities. Bacterial orthologs are phylogenetically divergent from eukaryotic NATs, and only a couple of them are characterized biochemically. Accordingly, not much is known about their substrate specificities. Rv3420c of Mycobacterium tuberculosis is a NAT ortholog coding for RimI(Mtb). Using in vitro peptide-based enzyme assays and mass-spectrometry methods, we provide evidence that RimI(Mtb) is a protein Nα-acetyltransferase of relaxed substrate specificity mimicking substrate specificities of eukaryotic NatA, NatC and most competently that of NatE. Also, hitherto unknown acetylation of residues namely, Asp, Glu, Tyr and Leu by a bacterial NAT (RimI(Mtb)) is elucidated, in vitro. Based on in vivo acetylation status, in vitro assay results and genetic context, a plausible cellular substrate for RimI(Mtb) is proposed.

Bacterial GCN5-Related N-Acetyltransferases: From Resistance to Regulation.

The GCN5-related N-acetyltransferases family (GNAT) is an important family of proteins that includes more than 100000 members among eukaryotes and prokaryotes. Acetylation appears as a major regulatory post-translational modification and is as widespread as phosphorylation. N-Acetyltransferases transfer an acetyl group from acetyl-CoA to a large array of substrates, from small molecules such as aminoglycoside antibiotics to macromolecules. Acetylation of proteins can occur at two different positions, either at the amino-terminal end (αN-acetylation) or at the ε-amino group (εN-acetylation) of an internal lysine residue. GNAT members have been classified into different groups on the basis of their substrate specificity, and in spite of a very low primary sequence identity, GNAT proteins display a common and conserved fold. This Current Topic reviews the different classes of bacterial GNAT proteins, their functions, their structural characteristics, and their mechanism of action.

Alba from Thermoplasma volcanium belongs to α-NAT's: An insight into the structural aspects of Tv Alba and its acetylation by Tv Ard1.

The Alba superfamily proteins have been regarded as a conserved group of proteins in archaea and eukarya, which have shown to be important in nucleic acid binding, chromatic organization and gene regulation. These proteins often belong to the N-acetyltransferase (NAT) category (N(α)-acetyltransferases or N(ε)-acetyltransferases) and undergo post-translational modifications. Here, we report the crystal structure of Alba from Thermoplasma volcanium (Tv Alba) at 2.4 Å resolution. The acetylation of Tv Alba was monitored and the N-terminal of Tv Alba has been shown to interact with acetyl coenzyme A (Ac-CoA). The chemical shift perturbation experiments of Tv Alba were performed in the presence of Ac-CoA and/or Tv Ard1, another T. volcanium protein that treats Tv Alba as a substrate. To examine the DNA binding capabilities of Tv Alba alone and in the presence of Ac-CoA and/or Tv Ard1, EMSA experiments were carried out. It is shown that although Tv Alba binds to Ac-CoA, the acetylation of Tv Alba is not related with its binding to dsDNA, and the involvement of the N-terminus in Ac-CoA binding demonstrates that Tv Alba belongs to the N(α)-acetyltransferase family.

Acetyl coenzyme A synthetase is acetylated on multiple lysine residues by a protein acetyltransferase with a single Gcn5-type N-acetyltransferase (GNAT) domain in Saccharopolyspora erythraea.

Reversible lysine acetylation (RLA) is used by cells of all domains of life to modulate protein function. To date, bacterial acetylation/deacetylation systems have been studied in a few bacteria (e.g., Salmonella enterica, Bacillus subtilis, Escherichia coli, Erwinia amylovora, Mycobacterium tuberculosis, and Geobacillus kaustophilus), but little is known about RLA in antibiotic-producing actinomycetes. Here, we identify the Gcn5-like protein acetyltransferase AcuA of Saccharopolyspora erythraea (SacAcuA, SACE_5148) as the enzyme responsible for the acetylation of the AMP-forming acetyl coenzyme A synthetase (SacAcsA, SACE_2375). Acetylated SacAcsA was deacetylated by a sirtuin-type NAD(+)-dependent consuming deacetylase (SacSrtN, SACE_3798). In vitro acetylation/deacetylation of SacAcsA enzyme was studied by Western blotting, and acetylation of lysine residues Lys(237), Lys(380), Lys(611), and Lys(628) was confirmed by mass spectrometry. In a strain devoid of SacAcuA, none of the above-mentioned Lys residues of SacAcsA was acetylated. To our knowledge, the ability of SacAcuA to acetylate multiple Lys residues is unique among AcuA-type acetyltransferases. Results from site-specific mutagenesis experiments showed that the activity of SacAcsA was controlled by lysine acetylation. Lastly, immunoprecipitation data showed that in vivo acetylation of SacAcsA was influenced by glucose and acetate availability. These results suggested that reversible acetylation may also be a conserved regulatory posttranslational modification strategy in antibiotic-producing actinomycetes.

In silico identification and characterization of N-Terminal acetyltransferase genes of poplar (Populus trichocarpa).

N-terminal acetyltransferase (Nats) complex is responsible for protein N-terminal acetylation (Nα-acetylation), which is one of the most common covalent modifications of eukaryotic proteins. Although genome-wide investigation and characterization of Nat catalytic subunits (CS) and auxiliary subunits (AS) have been conducted in yeast and humans they remain unexplored in plants. Here we report on the identification of eleven genes encoding eleven putative Nat CS polypeptides, and five genes encoding five putative Nat AS polypeptides in Populus. We document that the expansion of Nat CS genes occurs as duplicated blocks distributed across 10 of the 19 poplar chromosomes, likely only as a result of segmental duplication events. Based on phylogenetic analysis, poplar Nat CS were assigned to six subgroups, which corresponded well to the Nat CS types (CS of Nat A-F), being consistent with previous reports in humans and yeast. In silico analysis of microarray data showed that in the process of normal development of the poplar, their Nat CS and AS genes are commonly expressed at one relatively low level but share distinct tissue-specific expression patterns. This exhaustive survey of Nat genes in poplar provides important information to assist future studies on their functional role in poplar.

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

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.

Identification of the enzyme responsible for N-acetylation of norfloxacin by Microbacterium sp. Strain 4N2-2.

Microbacterium sp. 4N2-2, isolated from a wastewater treatment plant, converts the antibacterial fluoroquinolone norfloxacin to N-acetylnorfloxacin and three other metabolites. Because N-acetylation results in loss of antibacterial activity, identification of the enzyme responsible is important for understanding fluoroquinolone resistance. The enzyme was identified as glutamine synthetase (GS); N-acetylnorfloxacin was produced only under conditions associated with GS expression. The GS gene (glnA) was cloned, and the protein (53 kDa) was heterologously expressed and isolated. Optimal conditions and biochemical properties (K(m) and V(max)) of purified GS were characterized; the purified enzyme was inhibited by Mn(2+), Mg(2+), ATP, and ADP. The contribution of GS to norfloxacin resistance was shown by using a norfloxacin-sensitive Escherichia coli strain carrying glnA derived from Microbacterium sp. 4N2-2. The GS of Microbacterium sp. 4N2-2 was shown to act as an N-acetyltransferase for norfloxacin, which produced low-level norfloxacin resistance. Structural and docking analysis identified potential binding sites for norfloxacin at the ADP binding site and for acetyl coenzyme A (acetyl-CoA) at a cleft in GS. The results suggest that environmental bacteria whose enzymes modify fluoroquinolones may be able to survive in the presence of low fluoroquinolone concentrations.

Specificity and versatility of substrate binding sites in four catalytic domains of human N-terminal acetyltransferases.

Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs). In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p). To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate=MLG, EEE, MKG), hNaa10p/AcCoA/substrate (substrate=MLG, EEE). Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate's backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1' sites that is different for hNaa10p (acidic), hNaa20p (hydrophobic/basic), hNaa30p (basic) and hNaa50p (hydrophobic). We also observe dynamic correlation between the ligand binding site and helix [Formula: see text] that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions that should help rationalizing substrate-specificity and lay the ground for inhibitor design.