GCN5-related N-acetyltransferase Research Articles

Mechanism of translation inhibition by type II GNAT toxin AtaT2.

Type II toxin–antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin–antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin–antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 activity induces ribosome stalling at all four glycyl codons but does not evoke a stringent response. In vitro, AtaT2 acetylates the aminoacyl moiety of isoaccepting glycyl tRNAs, thus precluding their participation in translation. Our study broadens the known target specificity of GNAT toxins beyond the earlier described isoleucine and formyl methionine tRNAs, and suggest that various GNAT toxins may have evolved to specificaly target other if not all individual aminoacyl tRNAs.

Mistranslating tRNA identifies a deleterious S213P mutation in the Saccharomyces cerevisiaeeco1-1 allele.

Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial, with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within β7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored the function of the enzyme at elevated temperatures. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.

Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu.

The broad host range bacteriophage Mu employs a novel ‘methylcarbamoyl’ modification to protect its DNA from diverse restriction systems of its hosts. The DNA modification is catalyzed by a phage-encoded protein Mom, whose mechanism of action is a mystery. Here, we characterized the co-factor and metal-binding properties of Mom and provide a molecular mechanism to explain ‘methylcarbamoyl’ation of DNA by Mom. Computational analyses revealed a conserved GNAT (GCN5-related N-acetyltransferase) fold in Mom. We demonstrate that Mom binds to acetyl CoA and identify the active site. We discovered that Mom is an iron-binding protein, with loss of Fe2+/3+-binding associated with loss of DNA modification activity. The importance of Fe2+/3+ is highlighted by the colocalization of Fe2+/3+ with acetyl CoA within the Mom active site. Puzzlingly, acid-base mechanisms employed by >309,000 GNAT members identified so far, fail to support methylcarbamoylation of adenine using acetyl CoA. In contrast, free-radical chemistry catalyzed by transition metals like Fe2+/3+ can explain the seemingly challenging reaction, accomplished by collaboration between acetyl CoA and Fe2+/3+. Thus, binding to Fe2+/3+, a small but unprecedented step in the evolution of Mom, allows a giant chemical leap from ordinary acetylation to a novel methylcarbamoylation function, while conserving the overall protein architecture.

Encystment Induces Down-Regulation of an Acetyltransferase-Like Gene in Acanthamoeba castellanii.

Acanthamoeba castellanii is a ubiquitous free-living amoeba. Pathogenic strains are causative agents of Acanthamoeba keratitis and granulomatous amoebic encephalitis. In response to adverse conditions, A. castellanii differentiate into cysts, which are metabolically inactive and resistant cells. This process, also named encystment, involves biochemical and genetic modifications that remain largely unknown. This study characterizes the role of the ACA1_384820 Acanthamoeba gene during encystment. This gene encodes a putative N-acetyltransferase, belonging to the Gcn5-related N-acetyltransferase (GNAT) family. We showed that expression of the ACA1_384820 gene was down-regulated as early as two hours after induction of encystment in A. castellanii. Interestingly, overexpression of the ACA1_384820 gene affects formation of cysts. Unexpectedly, the search of homologs of ACA1_384820 in the Eukaryota gene datasets failed, except for some species in the Acanthamoeba genus. Bioinformatics analysis suggested a possible lateral acquisition of this gene from prokaryotic cells. This study enabled us to describe a new Acanthamoeba gene that is down-regulated during encystment.

A Conserved Tyrosine Residue of EcSpeG is Absolutely Critical for Enzymatic Activity

The Spermine/Spermidine N‐acetyltransferase from Escherichia coli (EcSpeG) is an allosteric dodecameric enzyme that belongs to the Gcn5‐related N‐acetyltransferase (GNAT) superfamily of enzymes. Under stressful conditions, SpeG acetylates the polyamines spermine (spm) and spermidine (spd), which bind to the active site and allosteric site of the protein. These polyamines are important polycationic molecules for many life processes including cell growth, protein synthesis, and metabolic regulation. GNATs commonly use a general acid base catalytic mechanism to acetylate their substrates and most have a key tyrosine residue that acts as the general acid in the reaction. It was previously shown that mutating this conserved residue in EcSpeG to phenylalanine did not abrogate the catalytic activity. In our experience, mutating this conserved residue in other GNATs has shown it is absolutely critical for activity. Therefore, we created multiple point mutations to test whether the identity of the residue at this position altered catalytic activity. Similar to our previous results with other GNATs, we found this tyrosine residue was critical for activity.Support or Funding InformationResearch reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).

Calcium Chloride Enhance Crystal Growth of an Uncharacterized GNAT Enzyme

Gcn5‐related N‐acetyltransferases (GNAT) are widespread across all domains of life and are capable of acetylating an assortment of substrates. However, many of their structures and functions remain unknown in bacteria. Our laboratory currently studies uncharacterized GNATs from Pseudomonas aeruginosa. We previously found the enzyme PA3944 acetylated polymyxin antibiotics and observed there was a duplicate GNAT gene (PA3945) with approximately 75% identity on the same operon. This protein has not been structurally or functionally characterized. Therefore, we screened this enzyme for activity using a broad‐substrate screening assay and set up crystallization screens to obtain protein crystals. Unfortunately, the PA3945 enzyme did not acetylate any substrates in the kinetic screening assay, which indicates it has a separate role compared to the PA3944 enzyme. We identified crystallization conditions that yielded microcrystals in the presence of calcium chloride. We further optimized these conditions using calcium chloride as an additive, which yielded larger crystals. Currently, we are investigating whether calcium chloride plays a role in enzyme activity and whether we can further optimize the crystallization conditions to yield suitable crystals for X‐ray diffraction and structure elucidation.Support or Funding InformationResearch reported in this work was supported by the National Science Foundation under Grant Number CHE‐1708863 (to MLK).

An Allosteric Site Residue Critical for Transmission of the Allosteric Signal of the Vibrio cholerae SpeG Enzyme

SpeG is a polyamine N‐acetyltransferase enzyme that belongs to the superfamily of Gcn5‐related N‐acetyltransferases (GNATs). We previously showed this enzyme from Vibrio cholerae (VcSpeG) acetylates spermine and spermidine. SpeG is thought to contribute to biofilm formation in several pathogenic bacteria and several studies have shown that it also plays a role in bacterial polyamine homeostasis under stressful conditions. This GNAT is unique because it forms a dodecameric structure and has an allosteric site in each monomer. Additionally, it binds polyamines at both allosteric and active sites. Previous X‐ray crystallographic studies have shown that the E41 residue in the allosteric site of VcSpeG forms a hydrogen bond with N10 of spermine and N6 of spermidine. We hypothesized that mutating this residue would affect the allosteric network of the enzyme. Therefore, we created a series of E41 point mutations and investigated the enzymatic activity and kinetic parameters toward spermine and spermidine. Changing the identity of the residue at this location either increased the catalytic efficiency and altered the sigmoidicity of the substrate saturation curve or decreased the catalytic efficiency and affected cooperativity. This implies that the identity of the residue at this position affects the allosteric signal that is transmitted. Further investigation into other residues in this site is warranted.Support or Funding InformationResearch reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).

Mapping Key Active Site Residues of the PA3944 Gcn5‐related N‐acetyltransferase

PA3944 is an acetyltransferase from Pseudomonas aeruginosa that acetylates polymyxin antibiotics in vitro. It belongs to the large Gcn5‐related N‐acetyltransferase (GNAT) superfamily. We previously determined the 3D structure of this enzyme, but were unable to obtain a complex in the presence of polymyxin. Since it is not know which residues in the active of this protein are critical for function and recognition of the polymyxin substrate, we created point mutations of residues in the active site and assayed their kinetic activity. We found two key residues that were critical for activity of the enyzme: one was important for the catalytic mechanism and the other played a structural role. Additionally, we found two mutants that improved the enzymatic activity, which could be used to further explore the possibility of using this enzyme for biotechnological applications to modify polymyxin antibiotics with reduced toxicity. Moreover, these results provide insight into GNAT evolution and how key residues can alter substrate specificity and structure.Support or Funding InformationResearch reported in this work was supported by the National Science Foundation under Grant Number CHE‐1708863 (to MLK).

Alternate fatty acid synthesis initiation in Escherichia coli

Fatty acid synthesis is important in both prokaryotes and eukaryotes for formation of functional membranes and dysregulation of the pathway has implications for cell growth and survival. In bacteria, fatty acid production is tightly regulated by the Type II fatty acid synthesis (FASII) pathway. FabH (3‐ketoacyl acyl carrier protein (ACP) synthase III) initiates fatty acid synthesis by condensing acetyl coenzyme A (acetyl‐CoA) with malonyl‐ACP in Escherichia coli. FabH was long thought to be the sole enzyme responsible for initiation of FASII and therefore essential for survival. The observation that fabH deletion strains are viable suggested the existence of an alternate initiation pathway. The yiiD gene was recently identified as essential for initiation of fatty acid synthesis in the absence of FabH in E. coli. The biochemical activity of YiiD is not known.YiiD has an N‐terminal GCN5‐related N‐acetyltransferase (GNAT) domain and a C‐terminal hotdog domain that is annotated as a thioesterase. The full‐length protein, as well as the N‐terminal and C‐terminal domains were purified using an E. coli expression system. Full length YiiD and the C‐terminal hotdog domain are dimers and the N‐terminal GNAT domain is a monomer.YiiD lacks condensing enzyme activity, suggesting that the initiation of FASII by YiiD is occurring through an alternate mechanism. We hypothesized that YiiD is initiating FASII by a transacylase reaction. FabH has acetyl‐CoA:ACP transacetylase activity, however protein extracts from an E. coli fabH deletion strain retain transacetylase activity. Purified YiiD has acetyl‐CoA:ACP transacetylase activity. The N‐terminal GNAT domain was inactive, whereas the C‐terminal domain was 100‐fold more active than intact YiiD. This suggests that the C‐terminal domain has the transacetylase active site and that the N‐terminal domain negatively regulates this activity. These data are consistent with YiiD forming acetyl‐ACP that is used by the elongation condensing enzymes (FabB or FabF) to initiate FASII thus bypassing FabH.Support or Funding InformationNational Institutes of Health Grant GM034496, Cancer Center (CORE) Support Grant CA21765, American and Lebanese Syrian Associated Charities (ALSAC)Proposed model for alternate initiation of fatty acid synthesis by YiiD in E. coliFigure 1

Repurposing the GNAT Fold in the Initiation of Polyketide Biosynthesis

Natural product biosynthetic pathways are replete with enzymes repurposed for new catalytic functions. In some modular polyketide synthase (PKS)pathways, a GCN5-related N-acetyltransferase (GNAT)-like enzyme with an additional decarboxylation function initiates biosynthesis. Here, we probe two PKS GNAT-like domains for the dual activities of S-acyl transfer from coenzyme A (CoA) to an acyl carrier protein (ACP) and decarboxylation. The GphF and CurA GNAT-like domains selectively decarboxylate substrates that yield the anticipated pathway starter units. The GphF enzyme lacks detectable acyl transfer activity, and a crystal structure with an isobutyryl-CoA product analog reveals a partially occluded acyltransfer acceptor site. Further analysis indicates that the CurA GNAT-like domain also catalyzes only decarboxylation, and the initial acyl transfer is catalyzed by an unidentified enzyme. Thus, PKS GNAT-like domains are re-classified as GNAT-like decarboxylases. Two other decarboxylases, malonyl-CoA decarboxylase and EryM, reside on distant nodes of the superfamily, illustrating the adaptability of the GNAT fold.

Molecular cloning and characterization of a grapevine (Vitis vinifera L.) serotonin N-acetyltransferase (VvSNAT2) gene involved in plant defense

BackgroundMelatonin is a ubiquitous molecule and exists across kingdoms. Studies on melatonin in plants have mainly focused on its physiological influence on growth and development, and on its biosynthesis. A number of studies have been conducted on the melatonin content and exogenous melatonin treatment of grapevine (Vitis vinifera L.). However, key genes or enzymes of the melatonin biosynthetic pathway remain unclear.ResultsIn this study, we cloned and identified the gene encoding serotonin N-acetyltransferase (SNAT) in grapevine (VvSNAT2). The VvSNAT2 protein was identified from a collection of 30 members of the grapevine GCN5-related N-acetyltransferase (GNAT) superfamily. Phylogenetic and protein sublocalization analyses showed that the candidate gene VvGNAT16 is VvSNAT2. Characterization of VvSNAT2 showed that its enzymatic activity is highest at a pH of 8.8 and a temperature of 45 °C. Analysis of enzyme kinetics showed the values of Km and Vmax of VvSNAT2 using serotonin were 392.5 μM and 836 pmol/min/mg protein, respectively. The expression of VvSNAT2 was induced by melatonin treatment and pathogen inoculation. Overexpression of VvSNAT2 in Arabidopsis resulted in greater accumulation of melatonin and chlorophyll and enhanced resistance to powdery mildew in the transgenic plants compared with the wild type (WT). Additionally, our data showed that the marker genes in the salicylic acid (SA) signaling pathway were expressed to higher levels in the transgenic plants compared with the WT.ConclusionsThe VvSNAT2 gene was cloned and identified in grapevine for the first time. Our results indicate that VvSNAT2 overexpression activates the SA and JA signaling pathways; however, the SA pathway plays a central role in VvSNAT2-mediated plant defense.

Elucidation of role of an acetyltransferase like protein in paromomycin resistance in Leishmania donovani using in silico and in vitro approaches

Paromomycin, an aminoglycoside antibiotic, is an effective treatment for VL (visceral leishmaniasis) in India. The modification of aminoglycoside antibiotics by enzymes such as aminoglycoside acetyltransferases is the predominant mechanism of resistance to antibiotics in bacterial system. In the present study, we identified and characterized LdATLP (an acetyltransferase-like protein) and elucidated its role in paromomycin resistance in Leishmania donovani. Gene encoding LdATLP was consistently up-regulated (>2fold) in three distinct paromomycin resistant in comparison with sensitive parasites, although the gene sequence was identical in the two. In silico analysis revealed that LdATLP consisted of conserved GNAT (GCN5-related N-Acetyltransferase) domain which is characteristic of aminoglycoside N-acetyltransferases. Evolutionary relationship among LdATLP of Leishmania and aminoglycoside acetyltransferases of bacteria was established by phylogenetic analysis. The 3D structure of LdATLP, predicted by ab-initio modeling, constituted 6 α-helices and 6 β-sheets. A few residues, such as R175, R177, E196, R197, V198, V200, K202, R205, C206, D208, G210, R211, R215, A234, S237, S238, K239, D240, F241 and Y242 of GNAT domain were predicted to be present at active site. Molecular docking of LdATLP with paromomycin or indolicidin (broad spectrum inhibitor of aminoglycoside modifying enzymes), followed by molecular dynamics simulation of docked complex suggested that both paromomycin and indolicidin bind to LdATLP with comparable free energy of binding. In vitro studies revealed that in the presence of indolicidin, paromomycin resistant parasites exhibited reversion of phenotype into sensitive parasites with marked increase in paromomycin susceptibility, suggesting the role of LdATLP in paromomycin resistance.Communicated by Ramaswamy H. Sarma

A MYST family histone acetyltransferase, MoSAS3, is required for development and pathogenicity in the rice blast fungus.

SummaryHistone acetylation has been established as a principal epigenetic regulatory mechanism in eukaryotes. Sas3, a histone acetyltransferase belonging to the largest family of acetyltransferase, MYST, is the catalytic subunit of a conserved histone acetyltransferase complex. To date, the functions of Sas3 and its orthologues have been extensively studied in yeast, humans and flies in relation to global acetylation and transcriptional regulation. However, its precise impact on development and pathogenicity in fungal plant pathogens has yet to be elucidated. Considering the importance of Sas3 in H3K14 acetylation, here we investigate the roles of its orthologue in the rice blast fungus, Magnaporthe oryzae (Pyricularia oryzae). Unlike a previously reported Sas3 deletion in yeast, which led to no remarkable phenotypic changes, we found that MoSAS3 deletion alone had a profound effect on fungal growth and development, including asexual reproduction, germination and appressorium formation in M. oryzae. Such defects in pre‐penetration development resulted in complete loss of pathogenicity in the deletion mutant. Furthermore, genetic analysis of MoSAS3 and MoGCN5 encoding a Gcn5‐related N‐acetyltransferase family histone acetyltransferase suggested that two conserved components of histone acetylation are integrated differently into epigenetic regulatory mechanisms in the yeast and a filamentous fungus. RNA‐seq analysis of ΔMosas3 showed two general trends: many DNA repair and DNA damage response genes are up‐regulated, while carbon and nitrogen metabolism genes are down‐regulated in ΔMosas3. Our work demonstrates the importance of MYST family histone acetyltransferase as a developmental regulator and illuminates a degree of functional variation in conserved catalytic subunits among different fungal species.

Toxin-antitoxin operon kacAT of Klebsiella pneumoniae is regulated by conditional cooperativity via a W-shaped KacA-KacT complex.

Bacterial toxin–antitoxin pairs play important roles in bacterial multidrug tolerance. Gcn5-related N-acetyltransferase (GNAT) toxins inhibit translation by acetylation of aminoacyl-tRNAs and are counteracted by direct contacts with cognate ribbon–helix–helix (RHH) antitoxins. Our previous analysis showed that the GNAT toxin KacT and RHH antitoxin KacA of Klebsiella pneumoniae form a heterohexamer in solution and that the complex interacts with the cognate promoter DNA, resulting in negative autoregulation of kacAT transcription. Here, we present the crystal structure of DNA-bound KacAT complex at 2.2 Å resolution. The crystal structure revealed the formation of a unique heterohexamer, KacT–KacA2–KacA2–KacT. The direct interaction of KacA and KacT involves a unique W-shaped structure with the two KacT molecules at opposite ends. Inhibition of KacT is achieved by the binding of four KacA proteins that preclude the formation of an active KacT dimer. The kacAT operon is auto-regulated and we present an experimentally supported molecular model proposing that the KacT:KacA ratio controls kacAT transcription by conditional cooperativity. These results yield a profound understanding of how transcription GNAT–RHH pairs are regulated.

Staphylococcus aureus modulates the activity of acetyl-Coenzyme A synthetase (Acs) by sirtuin-dependent reversible lysine acetylation.

Lysine acylation is a posttranslational modification used by cells of all domains of life to modulate cellular processes in response to metabolic stress. The paradigm for the role of lysine acylation in metabolism is the acetyl-coenzyme A synthetase (Acs) enzyme. In prokaryotic and eukaryotic cells alike, Acs activity is downregulated by acetylation and reactivated by deacetylation. Proteins belonging to the bacterial GCN5-related N-acetyltransferase (bGNAT) superfamily acetylate the epsilon amino group of an active site lysine, inactivating Acs. A deacetylase can remove the acetyl group, thereby restoring activity. Here we show the Acs from Staphylococcus aureus (SaAcs) activates acetate and weakly activates propionate, but does not activate >C3 organic acids or dicarboxylic acids (e.g. butyrate, malonate and succinate). SaAcs activity is regulated by AcuA (SaAcuA); a type-IV bGNAT. SaAcuA can acetylate or propionylate SaAcs reducing its activity by >90% and 95% respectively. SaAcuA also succinylated SaAcs, with this being the first documented case of a bacterial GNAT capable of succinylation. Inactive SaAcsAc was deacetylated (hence reactivated) by the NAD+ -dependent (class III) sirtuin protein deacetylase (hereafter SaCobB). In vivo and in vitro evidence show that SaAcuA and SaCobB modulate the level of SaAcs activity in S. aureus.

Complementary Roles of GCN5 and PCAF in Foxp3+ T-Regulatory Cells.

Functions of the GCN5-related N-acetyltransferase (GNAT) family of histone/protein acetyltransferases (HATs) in Foxp3+ T-regulatory (Treg) cells are unexplored, despite the general importance of these enzymes in cell biology. We now show that two prototypical GNAT family members, GCN5 (general control nonrepressed-protein 5, lysine acetyltransferase (KAT)2a) and p300/CBP-associated factor (p300/CBP-associated factor (PCAF), Kat2b) contribute to Treg functions through partially distinct and partially overlapping mechanisms. Deletion of Gcn5 or PCAF did not affect Treg development or suppressive function in vitro, but did affect inducible Treg (iTreg) development, and in vivo, abrogated Treg-dependent allograft survival. Contrasting effects were seen upon targeting of each HAT in all T cells; mice lacking GCN5 showed prolonged allograft survival, suggesting this HAT might be a target for epigenetic therapy in allograft recipients, whereas transplants in mice lacking PCAF underwent acute allograft rejection. PCAF deletion also enhanced anti-tumor immunity in immunocompetent mice. Dual deletion of GCN5 and PCAF led to decreased Treg stability and numbers in peripheral lymphoid tissues, and mice succumbed to severe autoimmunity by 3–4 weeks of life. These data indicate that HATs of the GNAT family have contributions to Treg function that cannot be replaced by the functions of previously characterized Treg HATs (CBP, p300, and Tip60), and may be useful targets in immuno-oncology.

Elucidating the Molecular Mechanisms of Antibiotic Resistance in Acinetobacter baumannii

The threat of antibiotic resistance has increasingly become a global issue because of the gradual emergence of multi‐drug resistant (MDR) pathogens. Identifying mechanisms of resistance as well as characterization of attributes deemed beneficial for the survival of the pathogen is vital in contributing to the development of new therapeutics. Acinetobacter baumannii is a gram‐negative opportunistic, nosocomial MDR pathogen that primarily targets immunocompromised patients in medical care facilities around the world. From an assembled bank of clinical isolates of A. baumannii, there is a diverse spectrum of antibiotic resistance patterns, cell motility and biofilm formation represented. Initial RNA sequencing studies implicated the importance of the Gcn5‐related N‐acetyltransferases (GNATs) in facilitating A baumannii resistance to the aminoglycoside class of antibiotics. We aim to determine the role of GNAT proteins to antibiotic resistance in A. baumannii as well as identify which physiological processes are associated with antibiotic resistance. GNATs belong to a superfamily of enzymes that are found in all domains of life and are involved various functions including acetylation of a broad spectrum of substrates and, often times, conferring resistance to antibiotics. We performed structural and functional experiments to understand the mechanisms causing the resistant nature of A. baumannii. Preliminary crystallization studies of the enzyme GNAT 2199 yielded 4 initial conditions suitable for optimal crystal growth. We also conducted a series of enzyme activity assays which revealed that GNATs acetylate various clinically used aminoglycosides. seeking to reveal the natural aminoglycoside substrate for GNAT 2199. Furthermore, cell motility and biofilm assays were used to determine the physiological processes attributed to antibiotic resistance of A. baumannii isolates. Future work entails optimization of the crystallization conditions to produce samples suitable for X‐ray diffraction studies. This will allow us to develop GNAT superfamily structure‐function relationships as well as identify physiological processes of A. baumannii that contribute to antibiotic resistance. In total, this information will be useful for initiating new strategies to control the spread of this bacteria as well as developing new therapeutics to effectively combat this MDR pathogen.Support or Funding InformationNSF No. HRD‐1547757This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanism of regulation and neutralization of the AtaR-AtaT toxin-antitoxin system.

GCN5-related N-acetyl-transferase (GNAT)-like enzymes from toxin-antitoxin modules are strong inhibitors of protein synthesis. Here, we present the bases of the regulatory mechanisms of ataRT, a model GNAT-toxin-antitoxin module, from toxin synthesis to its action as a transcriptional de-repressor. We show the antitoxin (AtaR) traps the toxin (AtaT) in a pre-catalytic monomeric state and precludes the effective binding of ac-CoA and its target Met-transfer RNAfMet. In the repressor complex, AtaR intrinsically disordered region interacts with AtaT at two different sites, folding into different structures, that are involved in two separate functional roles, toxin neutralization and placing the DNA-binding domains of AtaR in a binding-compatible orientation. Our data suggests AtaR neutralizes AtaT as a monomer, right after its synthesis and only the toxin-antitoxin complex formed in this way is an active repressor. Once activated by dimerization, later neutralization of the toxin results in a toxin-antitoxin complex that is not able to repress transcription.

Structural insights into the substrate binding mechanism of novel ArgA from Mycobacterium tuberculosis

The Mycobacterium tuberculosis (Mtb) Rv2747 gene encodes for a functional protein known as ArgA, which plays an important role in the first step of the l-arginine biosynthesis pathway. ArgA transfers the acetyl group from the acetyl-CoA to either l-glutamate or l-glutamine, which are the known substrates. Here, we present two crystal structures of ArgA: one complexed with CoA and product bound N-acetylglutamine and the other complexed with acetyl-CoA and the inhibitor l-arginine at 2.3 and 3.0 Å resolution respectively. The Mtb ArgA protomer was found to have a “V” cleft and a “β” bulge, archetypal of a classical GCN5-related N-acetyltransferase superfamily of proteins. The product bound form implies that ArgA can also acetylate l-glutamine like l-glutamate. The active site is strongly inhibited by l-arginine resulting in a closed conformation of ArgA and both l-arginine and N-acetylglutamine were found to occupy at the same active site. Together with structural analysis, molecular docking studies, microscale thermophoresis and enzyme inhibition assays, we conclude that l-glutamine, l-glutamate and l-arginine, all occupy at the same active site of ArgA. Furthermore in case of Mtb ArgA, l-arginine does not act as an allosteric inhibitor unlike other N-acetylglutamate synthase family of proteins.

P300/CBP-associated factor regulates transcription and function of isocitrate dehydrogenase 2 during muscle differentiation.

The epigenetic enzyme p300/CBP-associated factor (PCAF) belongs to the GCN5-related N-acetyltransferase (GNAT) family together with GCN5. Although its transcriptional and post-translational function is well characterized, little is known about its properties as regulator of cell metabolism. Here, we report the mitochondrial localization of PCAF conferred by an 85 aa mitochondrial targeting sequence (MTS) at the N-terminal region of the protein. In mitochondria, one of the PCAF targets is the isocitrate dehydrogenase 2 (IDH2) acetylated at lysine 180. This PCAF-regulated post-translational modification might reduce IDH2 affinity for isocitrate as a result of a conformational shift involving predictively the tyrosine at position 179. Site-directed mutagenesis and functional studies indicate that PCAF regulates IDH2, acting at dual level during myoblast differentiation: at a transcriptional level together with MyoD, and at a post-translational level by direct modification of lysine acetylation in mitochondria. The latter event determines a decrease in IDH2 function with negative consequences on muscle fiber formation in C2C12 cells. Indeed, a MTS-deprived PCAF does not localize into mitochondria, remains enriched into the nucleus, and contributes to a significant increase of muscle-specific gene expression enhancing muscle differentiation. The role of PCAF in mitochondria is a novel finding shedding light on metabolic processes relevant to early muscle precursor differentiation.-Savoia, M., Cencioni, C., Mori, M., Atlante, S., Zaccagnini, G., Devanna, P., Di Marcotullio, L., Botta, B., Martelli, F., Zeiher, A. M., Pontecorvi, A., Farsetti, A., Spallotta, F., Gaetano, C. P300/CBP-associated factor regulates transcription and function of isocitrate dehydrogenase 2 during muscle differentiation.