GCN5-related N-acetyltransferase Research Articles

MECHANISM OF DEPROTONATION OF THE AMINO GROUP OF GLUTAMATE UPON BINDING TO N-ACETYLGLUTAMATE SYNTHASE

Gcn5-related N-acetyltransferases catalyze the transfer of an acetyl group to a primary amino group of a wide class of substrates. Protonation of the amino group upon binding to the enzyme is necessary to activate the nucleophilic attack on the substrate. The process of glutamate binding to N-acetylglutamate synthase is considered using molecular modeling and quantum chemistry methods. It has been shown that deprotonation of the primary amino group of glutamate occurs upon binding to the active site of the enzyme with the participation of the side chain of the aspartate residue.

Molecular characterization and induced changes of histone acetyltransferases in the tick Haemaphysalis longicornis in response to cold stress

BackgroundEpigenetic modifications of histones play important roles in the response of eukaryotic organisms to environmental stress. However, many histone acetyltransferases (HATs), which are responsible for histone acetylation, and their roles in mediating the tick response to cold stress have yet to be identified. In the present study, HATs were molecularly characterized and their associations with the cold response of the tick Haemaphysalis longicornis explored.MethodsHATs were characterized by using polymerase chain reaction (PCR) based on published genome sequences, followed by multiple bioinformatic analyses. The differential expression of genes in H. longicornis under different cold treatment conditions was evaluated using reverse transcription quantitative PCR (RT-qPCR). RNA interference was used to explore the association of HATs with the cold response of H. longicornis.ResultsTwo HAT genes were identified in H. longicornis (Hl), a GCN5-related N-acetyltransferase (henceforth HlGNAT) and a type B histone acetyltransferase (henceforth HlHAT-B), which are respectively 960 base pairs (bp) and 1239 bp in length. Bioinformatics analysis revealed that HlGNAT and HlHAT-B are unstable hydrophilic proteins characterized by the presence of the acetyltransferase 16 domain and Hat1_N domain, respectively. RT-qPCR revealed that the expression of HlGNAT and HlHAT-B decreased after 3 days of cold treatment, but gradually increased with a longer period of cold treatment. The mortality rate following knockdown of HlGNAT or HlHAT-B by RNA interference, which was confirmed by RT-qPCR, significantly increased (P < 0.05) when H. longicornis was treated at the lowest lethal temperature (− 14 °C) for 2 h.ConclusionsThe findings demonstrate that HATs may play a crucial role in the cold response of H. longicornis. Thus further research is warranted to explore the mechanisms underlying the epigenetic regulation of the cold response in ticks.Graphical

Characterization of an agmatine N-acetyltransferase from Bactrocera dorsalis that modulates ovary development

Agmatine N-acetyltransferase (AgmNAT), which catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine, is a member of the GCN5-related N-acetyltransferase family. So far, knowledge of the physiological roles of AgmNAT in insects is limited. Here, we identified one gene encoding protein homologous to that of Drosophila AgmNAT using sequence information from an activity-verified Drosophila AgmNAT in a BLAST search of the Bactrocera dorsalis genome. We expressed and purified B. dorsalis AgmNAT in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Our application of the screening strategy to BdorAgmNAT led to the identification of agmatine as the best amine substrate for this enzyme, with the highest kcat/Km value. We successfully obtained a BdorAgmNAT knockout strain based on a wild-type strain (WT) using the CRISPR/Cas9 technique. The ovary development of the BdorAgmNAT knockout mutants was delayed for 10 days compared with the WT specimens. Moreover, mutants had a much smaller mature ovary size and laid far fewer eggs than WT. Loss of function of BdorAgmNAT caused by RNAi with mature WT females did not affect their fecundity. These findings indicate that BdorAgmNAT is critical for oogenesis. Our data provide the first evidence for AgmNAT in regulating ovary development.

An acetyltransferase effector conserved across Legionella species targets the eukaryotic eIF3 complex to modulate protein translation.

The survival of Legionella spp. as intracellular pathogens relies on the combined action of protein effectors delivered inside their eukaryotic hosts by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IVb secretion system. The specific repertoire of effector arsenals varies dramatically across over 60 known species of this genera with Legionella pneumophila responsible for most cases of Legionnaires' disease in humans encoding over 360 Dot/Icm effectors. However, a small subset of "core" effectors appears to be conserved across all Legionella species raising an intriguing question of their role in these bacteria's pathogenic strategy, which for most of these effectors remains unknown. L. pneumophila Lpg0103 effector, also known as VipF, represents one of the core effector families that features a tandem of Gcn5-related N-acetyltransferase (GNAT) domains. Here, we present the crystal structure of the Lha0223, the VipF representative from Legionella hackeliae in complex with acetyl-coenzyme A determined to 1.75 Å resolution. Our structural analysis suggested that this effector family shares a common fold with the two GNAT domains forming a deep groove occupied by residues conserved across VipF homologs. Further analysis suggested that only the C-terminal GNAT domain of VipF effectors retains the active site composition compatible with catalysis, whereas the N-terminal GNAT domain binds the ligand in a non-catalytical mode. We confirmed this by in vitro enzymatic assays which revealed VipF activity not only against generic small molecule substrates, such as chloramphenicol, but also against poly-L-lysine and histone-derived peptides. We identified the human eukaryotic translation initiation factor 3 (eIF3) complex co-precipitating with Lpg0103 and demonstrated the direct interaction between the several representatives of the VipF family, including Lpg0103 and Lha0223 with the K subunit of eIF3. According to our data, these interactions involve primarily the C-terminal tail of eIF3-K containing two lysine residues that are acetylated by VipF. VipF catalytic activity results in the suppression of eukaryotic protein translation in vitro, revealing the potential function of VipF "core" effectors in Legionella's pathogenic strategy.IMPORTANCEBy translocating effectors inside the eukaryotic host cell, bacteria can modulate host cellular processes in their favor. Legionella species, which includes the pneumonia-causing Legionella pneumophila, encode a widely diverse set of effectors with only a small subset that is conserved across this genus. Here, we demonstrate that one of these conserved effector families, represented by L. pneumophila VipF (Lpg0103), is a tandem Gcn5-related N-acetyltransferase interacting with the K subunit of human eukaryotic initiation factor 3 complex. VipF catalyzes the acetylation of lysine residues on the C-terminal tail of the K subunit, resulting in the suppression of eukaryotic translation initiation factor 3-mediated protein translation in vitro. These new data provide the first insight into the molecular function of this pathogenic factor family common across Legionellae.

An acetyltranferase moonlights as a regulator of the RNA binding repertoire of the RNA chaperone Hfq in Escherichia coli.

Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, a mediator for sRNA-mRNA annealing. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was previously identified as a silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and investigated its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppressed all tested Hfq-dependent sRNA signaling. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked the interaction interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, and HqbA interacted with Hfq at a site far from the active site. This suggests that HqbA may be bifunctional, with separate roles for regulating via Hfq interaction and for acetylation of undefined substrates. Gel shift assays revealed that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs but not high-affinity RNAs. Comparative RNA immunoprecipitation of Hfq and sequencing showed enrichment of two tRNA precursors, metZWV and proM, by Hfq in mutants that lost the HqbA-Hfq interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.

Deciphering the structure, function, and mechanism of lysine acetyltransferase cGNAT2 in cyanobacteria.

Lysine acetylation is a conserved regulatory posttranslational protein modification that is performed by lysine acetyltransferases (KATs). By catalyzing the transfer of acetyl groups to substrate proteins, KATs play critical regulatory roles in all domains of life; however, no KATs have yet been identified in cyanobacteria. Here, we tested all predicted KATs in the cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) and demonstrated that A1596, which we named cyanobacterial Gcn5-related N-acetyltransferase (cGNAT2), can catalyze lysine acetylation in vivo and in vitro. Eight amino acid residues were identified as the key residues in the putative active site of cGNAT2, as indicated by structural simulation and site-directed mutagenesis. The loss of cGNAT2 altered both growth and photosynthetic electron transport in Syn7002. In addition, quantitative analysis of the lysine acetylome identified 548 endogenous substrates of cGNAT2 in Syn7002. We further demonstrated that cGNAT2 can acetylate NAD(P)H dehydrogenase J (NdhJ) in vivo and in vitro, with the inability to acetylate K89 residues, thus decreasing NdhJ activity and affecting both growth and electron transport in Syn7002. In summary, this study identified a KAT in cyanobacteria and revealed that cGNAT2 regulates growth and photosynthesis in Syn7002 through an acetylation-mediated mechanism.

The Brucella Effector Protein BspF Regulates Apoptosis through the Crotonylation of p53.

The Brucella type IV secretion system (T4SS) can promote the intracellular survival and reproduction of Brucella. T4SS secretes effector proteins to act on cellular signaling pathways to inhibit the host's innate immune response and cause a chronic, persistent Brucella infection. Brucella can survive in host cells for a long time by inhibiting macrophage apoptosis and avoiding immune recognition. The effector protein, BspF, secreted by T4SS, can regulate host secretory transport and accelerate the intracellular replication of Brucella. BspF has an acetyltransferase domain of the GNAT (GCN5-related N-acetyltransferases) family, and in our previous crotonylation proteomics data, we have found that BspF has crotonyl transferase activity and crotonylation regulation of host cell protein in the proteomics data. Here, we found that BspF attenuates the crotonylation modification of the interacting protein p53, which reduces the p53 expression through the GNAT domain. BspF can inhibit the transcription and protein expression of downstream apoptotic genes, thereby inhibiting host cell apoptosis. Additionally, the Brucella ΔbspF mutant stain promotes apoptosis and reduces the survival rate of Brucella in the cells. In conclusion, we identified that the T4SS effector protein BspF can regulate host cell apoptosis to assist Brucella in its long-term survival by attenuating crotonylation modification of p53 and decreasing p53 expression. Our findings reveal a unique mechanism of elucidating how Brucella regulates host cell apoptosis and promotes its proliferation through the secretion of effector proteins.

Lysine lactylation regulates metabolic pathways and biofilm formation in Streptococcus mutans.

In eukaryotes, lactate produced during glycolysis is involved in regulating multiple metabolic processes through lysine lactylation (Kla). To explore the potential link between metabolism and Kla in prokaryotes, we investigated the distribution of Kla in the cariogenic bacterium Streptococcus mutans during planktonic growth in low-sugar conditions and in biofilm-promoting, high-sugar conditions. We identified 1869 Kla sites in 469 proteins under these two conditions, with the biofilm growth state showing a greater number of lactylated sites and proteins. Although high sugar increased Kla globally, it reduced lactylation of RNA polymerase subunit α (RpoA) at Lys173. Lactylation at this residue inhibited the synthesis of extracellular polysaccharides, a major constituent of the cariogenic biofilm. The Gcn5-related N-acetyltransferase (GNAT) superfamily enzyme GNAT13 exhibited lysine lactyltransferase activity in cells and lactylated Lys173 in RpoA in vitro. Either GNAT13 overexpression or lactylation of Lys173 in RpoA inhibited biofilm formation. These results provide an overview of the distribution and potential functions of Kla and improve our understanding of the role of lactate in the metabolic regulation of prokaryotes.

Crystal structure of a GCN5-related N-acetyltransferase from Lactobacillus curiae.

Members of the GCN5-related N-acetyltransferase (GNAT) family are found in all domains of life and are involved in processes ranging from protein synthesis and gene expression to detoxification and virulence. Due to the variety of their macromolecular targets, GNATs are a highly diverse family of proteins. Currently, 3D structures of only a small number of GNAT representatives are available and thus the family remains poorly characterized. Here, the crystal structure of the guanidine riboswitch-associated GNAT from Lactobacillus curiae (LcGNAT) that acetylates canavanine, a structural analogue of arginine with antimetabolite properties, is reported. LcGNAT shares the conserved fold of the members of the GNAT superfamily, but does not contain an N-terminal β0 strand and instead contains a C-terminal β7 strand. Its P-loop, which coordinates the pyrophosphate moiety of the acetyl-coenzyme A cosubstrate, is degenerated. These features are shared with its closest homologues in the polyamine acetyltransferase subclass. Site-directed mutagenesis revealed a central role of the conserved residue Tyr142 in catalysis, as well as the semi-conserved Tyr97 and Glu92, suggesting that despite its individual substrate specificity LcGNAT performs the classical reaction mechanism of this family.

Mapping roles of active site residues in the acceptor site of the PA3944 Gcn5-related N-acetyltransferase (GNAT) enzyme.

An increased understanding of how the acceptor site in Gcn5-related N-acetyltransferase (GNAT) enzymes recognizes various substrates provides important clues for GNAT functional annotation and their use as chemical tools. In this study, we explored how the PA3944 enzyme from Pseudomonas aeruginosa recognizes three different acceptor substrates, including aspartame, NANMO, and polymyxin B, and identified acceptor residues that are critical for substrate specificity. To achieve this, we performed a series of molecular docking simulations and tested methods to identify acceptor substrate binding modes that are catalytically relevant. We found that traditional selection of best docking poses by lowest S scores did not reveal acceptor substrate binding modes that were generally close enough to the donor for productive acetylation. Instead, sorting poses based on distance between the acceptor amine nitrogen atom and donor carbonyl carbon atom placed these acceptor substrates near residues that contribute to substrate specificity and catalysis. To assess whether these residues are indeed contributors to substrate specificity, we mutated seven amino acid residues to alanine and determined their kinetic parameters. We identified several residues that improved the apparent affinity and catalytic efficiency of PA3944, especially for NANMO and/or polymyxin B. Additionally, one mutant (R106A) exhibited substrate inhibition toward NANMO, and we propose scenarios for the cause of this inhibition based on additional substrate docking studies with R106A. Ultimately, we propose that this residue is a key gatekeeper between the acceptor and donor sites by restricting and orienting the acceptor substrate within the acceptor site. This article is protected by copyright. All rights reserved.

Acetylation of inorganic pyrophosphatase by S-RNase signaling induces pollen tube tip swelling by repressing pectin methylesterase.

Self-incompatibility (SI) is a widespread genetically determined system in flowering plants that prevents self-fertilization to promote gene flow and limit inbreeding. S-RNase-based SI is characterized by the arrest of pollen tube growth through the pistil. Arrested pollen tubes show disrupted polarized growth and swollen tips, but the underlying molecular mechanism is largely unknown. Here, we demonstrate that the swelling at the tips of incompatible pollen tubes in pear (Pyrus bretschneideri [Pbr]) is mediated by the SI-induced acetylation of the soluble inorganic pyrophosphatase (PPA) PbrPPA5. Acetylation at Lys-42 of PbrPPA5 by the acetyltransferase GCN5-related N-acetyltransferase 1 (GNAT1) drives accumulation of PbrPPA5 in the nucleus, where it binds to the transcription factor PbrbZIP77, forming a transcriptional repression complex that inhibits the expression of the pectin methylesterase (PME) gene PbrPME44. The function of PbrPPA5 as a transcriptional repressor does not require its PPA activity. Downregulating PbrPME44 resulted in increased levels of methyl-esterified pectins in growing pollen tubes, leading to swelling at their tips. These observations suggest a mechanism for PbrPPA5-driven swelling at the tips of pollen tubes during the SI response. The targets of PbrPPA5 include genes encoding cell wall-modifying enzymes, which are essential for building a continuous sustainable mechanical structure for pollen tube growth.

Molecular Cloning and Characterization of a Serotonin N-Acetyltransferase Gene, xoSNAT3, from Xanthomonas oryzae pv. oryzae.

Rice bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the top ten bacterial plant diseases worldwide. Serotonin N-acetyltransferase (SNAT) is one of the key rate-limiting enzymes in melatonin (MT) biosynthesis. However, its function in pathogenic bacteria remains unclear. In this study, a Xoo SNAT protein (xoSNAT3) that showed 27.39% homology with sheep SNAT was identified from a collection of 24 members of GCN5-related N-acetyltransferase (GNAT) superfamily in Xoo. This xoSNAT3 could be induced by MT. In tobacco-based transient expression system, xoSNAT3 was found localized on mitochondria. In vitro studies indicated that xoSNAT3 showed the optima enzymatic activity at 50 °C. The recombinant enzyme showed Km and Vmax values of 709.98 μM and 2.21 nmol/min/mg protein, respectively. Mutant △xoSNAT3 showed greater impaired MT biosynthesis than the wild-type strain. Additionally, △xoSNAT3 showed 14.06% less virulence and 26.07% less biofilm formation. Collectively, our results indicated that xoSNAT3 services as a SNAT involved in MT biosynthesis and pathogenicity in Xoo.

Function, structure, evolution, regulation of a potent drug target, arylalkylamine N-acetyltransferase.

Arylalkylamine N-acetyltransferase (aaNAT) catalyzes the transacetylation of acetyl coenzyme A to arylamines and arylalkylamines. Based on three-dimensional structural information, aaNAT belongs to the GCN5-related N-acetyltransferases superfamily with a conserved acetyl-CoA binding domain (Dyda et al., 2000). By comparison of sequence similarity, aaNAT is usually divided into vertebrate aaNAT (VT-aaNAT) and non-vertebrate aaNAT (NV-aaNAT) (Cazaméa-Catalan et al., 2014). Insects have evolved multiple aaNATs in comparison to mammals, thus more diverse functions are also reflected in insects. This chapter will summarize previous studies on the function, regulation, structure and evolution of aaNAT, and provide insight into future pest management.

The novel type II toxin-antitoxin PacTA modulates Pseudomonas aeruginosa iron homeostasis by obstructing the DNA-binding activity of Fur.

Type II toxin–antitoxin (TA) systems are widely distributed in bacterial and archaeal genomes and are involved in diverse critical cellular functions such as defense against phages, biofilm formation, persistence, and virulence. GCN5-related N-acetyltransferase (GNAT) toxin, with an acetyltransferase activity-dependent mechanism of translation inhibition, represents a relatively new and expanding family of type II TA toxins. We here describe a group of GNAT-Xre TA modules widely distributed among Pseudomonas species. We investigated PacTA (one of its members encoded by PA3270/PA3269) from Pseudomonas aeruginosa and demonstrated that the PacT toxin positively regulates iron acquisition in P. aeruginosa. Notably, other than arresting translation through acetylating aminoacyl-tRNAs, PacT can directly bind to Fur, a key ferric uptake regulator, to attenuate its DNA-binding affinity and thus permit the expression of downstream iron-acquisition-related genes. We further showed that the expression of the pacTA locus is upregulated in response to iron starvation and the absence of PacT causes biofilm formation defect, thereby attenuating pathogenesis. Overall, these findings reveal a novel regulatory mechanism of GNAT toxin that controls iron-uptake-related genes and contributes to bacterial virulence.

The Structure of Saccharomyces cerevisiae Arginyltransferase 1 (ATE1)

Eukaryotic post-translational arginylation, mediated by the family of enzymes known as the arginyltransferases (ATE1s), is an important post-translational modification that can alter protein function and even dictate cellular protein half-life. Multiple major biological pathways are linked to the fidelity of this process, including neural and cardiovascular developments, cell division, and even the stress response. Despite this significance, the structural, mechanistic, and regulatory mechanisms that govern ATE1 function remain enigmatic. To that end, we have used X-ray crystallography to solve the crystal structure of ATE1 from the model organism Saccharomyces cerevisiae ATE1 (ScATE1) in the apo form. The three-dimensional structure of ScATE1 reveals a bilobed protein containing a GCN5-related N-acetyltransferase (GNAT) fold, and this crystalline behavior is faithfully recapitulated in solution based on size-exclusion chromatography-coupled small angle X-ray scattering (SEC-SAXS) analyses and cryo-EM 2D class averaging. Structural superpositions and electrostatic analyses point to this domain and its domain-domain interface as the location of catalytic activity and tRNA binding, and these comparisons strongly suggest a mechanism for post-translational arginylation. Additionally, our structure reveals that the N-terminal domain, which we have previously shown to bind a regulatory [Fe-S] cluster, is dynamic and disordered in the absence of metal bound in this location, hinting at the regulatory influence of this region. When taken together, these insights bring us closer to answering pressing questions regarding the molecular-level mechanism of eukaryotic post-translational arginylation.

Biochemical, structural, and functional studies reveal that MAB_4324c from Mycobacterium abscessus is an active tandem repeat N-acetyltransferase.

Mycobacterium abscessus is a pathogenic non-tuberculous mycobacterium that possesses an intrinsic drug resistance profile. Several N-acetyltransferases mediate drug resistance and/or participate in M. abscessus virulence. Mining the M. abscessus genome has revealed genes encoding additional N-acetyltransferases whose functions remain uncharacterized, among them MAB_4324c. Here, we showed that the purified MAB_4324c protein is a N-acetyltransferase able to acetylate small polyamine substrates. The crystal structure of MAB_4324c was solved at high resolution in complex with its cofactor, revealing the presence of two GCN5-related N-acetyltransferase domains and a cryptic binding site for NADPH. Genetic studies demonstrate that MAB_4324c is not essential for in vitro growth of M. abscessus; however, overexpression of the protein enhanced the uptake and survival of M. abscessus in THP-1 macrophages.

Biosynthesis of Argolaphos Illuminates the Unusual Biochemical Origins of Aminomethylphosphonate and Nε-Hydroxyarginine Containing Natural Products.

Phosphonate natural products have a history of successful application in medicine and biotechnology due to their ability to inhibit essential cellular pathways. This has inspired efforts to discover phosphonate natural products by prioritizing microbial strains whose genomes encode uncharacterized biosynthetic gene clusters (BGCs). Thus, success in genome mining is dependent on establishing the fundamental principles underlying the biosynthesis of inhibitory chemical moieties to facilitate accurate prediction of BGCs and the bioactivities of their products. Here, we report the complete biosynthetic pathway for the argolaphos phosphonopeptides. We uncovered the biochemical origins of aminomethylphosphonate (AMPn) and Nε-hydroxyarginine, two noncanonical amino acids integral to the antimicrobial function of argolaphos. Critical to this pathway were dehydrogenase and transaminase enzymes dedicated to the conversion of hydroxymethylphosphonate to AMPn. The interconnected activities of both enzymes provided a solution to overcome unfavorable energetics, empower cofactor regeneration, and mediate intermediate toxicity during these transformations. Sequential ligation of l-arginine and l-valine was afforded by two GCN5-related N-acetyltransferases in a tRNA-dependent manner. AglA was revealed to be an unusual heme-dependent monooxygenase that hydroxylated the Nε position of AMPn-Arg. As the first biochemically characterized member of the YqcI/YcgG protein family, AglA enlightens the potential functions of this elusive group, which remains biochemically distinct from the well-established P450 monooxygenases. The widespread distribution of AMPn and YqcI/YcgG genes among actinobacterial genomes suggests their involvement in diverse metabolic pathways and cellular functions. Our findings illuminate new paradigms in natural product biosynthesis and realize a significant trove of AmPn and Nε-hydroxyarginine natural products that await discovery.

Characterization of Enzymes Catalyzing the Initial Steps of the β-Lactam Tabtoxin Biosynthesis

Tabtoxin is a β-lactam ring-containing phytotoxin produced by a plant pathogenic Pseudomonas species. Here, we describe the early stages of tabtoxin biosynthesis, involving a C-methylation reaction catalyzed by the S-adenosyl-l-methionine-dependent methyltransferase TblA as the initial step for the β-lactam construction. Gene deletion and in vitro biochemical assays demonstrated that the Gcn5-related N-acetyltransferase domain of TblD catalyzes the acetylation of the α-amino group of 5-methyl-l-lysine. This establishment of the early reaction steps lays the foundation for characterizing unique β-lactam biosynthesis.

Structural investigations of arginyltransferases

Arginyltransferases (ATE1s) are a class of essential eukaryotic enzymes that catalyze arginylation, the post‐translational transfer of Arg from an aminoacylated tRNA to a range of protein targets. This modification typically occurs at N‐terminal acidic residues, though ATE1 can also covalently attach Arg to mid‐chain residues. Arginylation may have either degradative or non‐degradative effects. For example, some arginylated proteins are processed through the Arg N‐degron pathway, which marks these post‐translationally modified proteins for degradation by the ubiquitin‐proteasome system. In contrast, arginylation may also manifest non‐degradative effects such as thermodynamic stability, subcellular relegalization, or functional changes. The diversity of ATE1’s targets confers its role as a global regulator, influencing functions such as cardiovascular development, neurological processing, and even the stress response. However, a lack of ATE1 structural knowledge has limited the determination of its three‐dimensional fold, how it is regulated, and how it recognizes its substrates. Using a combination of X‐ray crystallography, cryo‐EM, and size‐exclusion chromatography‐coupled small angle X‐ray scattering (SEC‐SAXS), our lab has successfully solved the structure of Saccharomyces cerevisiae ATE1 (ScATE1). The three‐dimensional structure of ScATE1 reveals a bilobed protein containing a canonical GCN5‐related N‐acetyltransferase (GNAT) fold. Structural superpositions and electrostatic analyses indicate this domain as the location of catalytic activity and tRNA binding. Furthermore, the structure reveals the spatial connectivity of the N‐terminal domain, which we previously showed binds an [Fe‐S] cluster, to the enzymatic active site, hinting at the cluster’s regulatory influence. As the first atomic‐level structure of any ATE1, this achievement brings us closer to answering pressing questions regarding the molecular‐level mechanism of eukaryotic post‐translational arginylation.

Structural and regulatory elements of post‐translational arginylation

Eukaryotic post‐translational arginylation, mediated by the family of enzymes known as the arginine transferases (ATE1s), is an important post‐translational modification that can alter protein function and even dictate cellular protein half‐life. Multiple major biological pathways are linked to the fidelity of this process, including neural and cardiovascular developments, cellular division, cytoskeletal function, and even the stress response. Despite this significance, the structural, mechanistic, and regulatory mechanisms that govern ATE1 function remain enigmatic. While exploring arginine transferase function, we have discovered that ATE1s bind a previously undiscovered [Fe‐S] cluster. We have used biochemical, spectroscopic, and analytical methods to decipher the composition and reactivity of this [Fe‐S] cluster. Fascinatingly, we find that ATE1 cluster‐binding preserves oligomeric homogeneity while increasing arginylation efficacy, demonstrating that this evolutionarily‐conserved [Fe‐S] cluster regulates arginylation rates. Furthermore, using a combination of X‐ray crystallography and size‐exclusion chromatography‐coupled small angle X‐ray scattering (SEC‐SAXS), we have successfully solved the first atomic‐level structure of an ATE1, which favorably recapitulates its solution‐state behavior. The three‐dimensional structure of ATE1 reveals a bilobed protein containing a GCN5‐related N‐acetyltransferase (GNAT) fold. Structural superpositions, peptide binding, and electrostatic analyses indicate this domain as the location of catalytic arginylation. Additionally, our structure reveals the spatial connectivity of the catalytic machinery to the N‐terminal domain that binds the [Fe‐S] cluster, hinting at the atomic‐level details of the cluster’s regulatory influence. Coupled with a new regulatory framework, the first atomic‐level structure of any ATE1 brings us closer to answering pressing questions regarding the molecular‐level mechanism of eukaryotic post‐translational arginylation.