Association of N-acetyltransferases 1 and 2 Polymorphisms with Susceptibility to Head and Neck Cancers-A Meta-Analysis, Meta-Regression, and Trial Sequential Analysis.

Background and objective:N-acetyltransferases 1 and 2 (NAT1 and NAT2) genes have polymorphisms in accordance with slow and rapid acetylator phenotypes with a role in the development of head and neck cancers (HNCs). Herein, we aimed to evaluate the association of NAT1 and NAT2 polymorphisms with susceptibility to HNCs in an updated meta-analysis. Materials and methods: A search was comprehensively performed in four databases (Web of Science, Scopus, PubMed/Medline, and Cochrane Library until 8 July 2021). The effect sizes, odds ratio (OR) along with 95% confidence interval (CI) were computed. Trial sequential analysis (TSA), publication bias and sensitivity analysis were conducted. Results: Twenty-eight articles including eight studies reporting NAT1 polymorphism and twenty-five studies reporting NAT2 polymorphism were involved in the meta-analysis. The results showed that individuals with slow acetylators of NAT2 polymorphism are at higher risk for HNC OR: 1.22 (95% CI: 1.02, 1.46; p = 0.03). On subgroup analysis, ethnicity, control source, and genotyping methods were found to be significant factors in the association of NAT2 polymorphism with the HNC risk. TSA identified that the amount of information was not large enough and that more studies are needed to establish associations. Conclusions: Slow acetylators in NAT2 polymorphism were related to a high risk of HNC. However, there was no relationship between NAT1 polymorphism and the risk of HNC.

From transglutaminases (TGs) to arylamine N-acetyltransferases (NATs): Insight into the role of a spatially conserved aromatic amino acid position in the active site of these two families of enzymes.

Transglutaminases (TG) and arylamine N-acetyltransferases (NAT) are important family of enzymes. Although they catalyze different reactions and have distinct structures, these two families of enzymes share a spatially conserved catalytic triad (Cys, His, Asp residues). In active TGs, a conserved Trp residue located close to the triad cysteine is crucial for catalysis through stabilization of transition states. Here, we show that in addition to sharing a similar catalytic triad with TGs, functional NAT enzymes also possess in their active site an aromatic residue (Phe, Tyr or Trp) occupying a structural position similar to the Trp residue of active TGs. More importantly, as observed in active TGs, our data indicates that in functional NAT enzymes this conserved aromatic residue is also involved in stabilization of transition states. These results thus indicate that in addition to the three triad residues, these two families of enzymes also share a spatially conserved aromatic amino acid position important for catalysis. Identification of residues involved in the stabilization of transition states is important to develop potent inhibitors. Interestingly, NAT enzymes have been shown as potential targets of clinical interest.

Slow N-acetylation as a possible contributor to bladder carcinogenesis.

Bladder cancer (BCa) is an exophytic tumor that presents as either noninvasive confined to the mucosa (NMIBC) or invading the detrusor muscle (MIBC), and was recently further subgrouped into molecular subtypes. Arylamines, major BCa environmental and occupational risk factors, are mainly metabolized by the genetically polymorphic N-acetyltransferases 1, NAT1 and NAT2. In this study, we investigated the association between N-acetyltransferases genetic polymorphism and key MIBC and NMIBC tumor biomarkers and subtypes. A cohort of 250 males with histologically confirmed urothelial BCa was identified. Tumors were genotyped for NAT1 and NAT2 using real-time polymerase chain reaction (PCR), and characterized for mutations in TP53, RB1, and FGFR3 by PCR-restriction fragment length polymorphism. Pathology data and patients' smoking status were obtained from medical records. Pearson χ2 and Fisher exact tests were used to check for associations and interactions. Results show that NAT1 G560 A polymorphism is significantly associated with higher muscle-invasiveness (MIBC vs NMIBC; P = .001), higher tumor grade (high grade vs low grade; P = .011), and higher FGFR3 mutation frequency within the MIBC subgroup (P = .042; .027). NAT2 G857 A polymorphism is also found to be significantly associated with higher muscle-invasiveness (MIBC vs NMIBC; P = .041). Our results indicate that slow N-acetylation is a contributor to bladder carcinogenesis and muscle-invasiveness. These findings highlight NAT1 as a biomarker candidate in BCa and a potential target for drug development.

Unexpected Acetylation of Endogenous Aliphatic Amines by Arylamine N-Acetyltransferase NAT2.

N-Acetyltransferases play critical roles in the deactivation and clearance of xenobiotics, including clinical drugs. NAT2 has been classified as an arylamine N-acetyltransferase that mainly converts aromatic amines, hydroxylamines, and hydrazines. Herein, we demonstrate that the human arylamine N-acetyltransferase NAT2 also acetylates aliphatic endogenous amines. Metabolomic analysis and chemical synthesis revealed increased intracellular concentrations of mono- and diacetylated spermidine in human cell lines expressing the rapid compared to the slow acetylator NAT2 phenotype. The regioselective N8 -acetylation of monoacetylated spermidine by NAT2 answers the long-standing question of the source of diacetylspermidine. We also identified selective acetylation of structurally diverse alkylamine-containing drugs by NAT2, which may contribute to variations in patient responses. The results demonstrate a previously unknown functionality and potential regulatory role for NAT2, and we suggest that this enzyme should be considered for re-classification.

Insights into the Function of the N-Acetyltransferase SatA That Detoxifies Streptothricin in Bacillus subtilis and Bacillus anthracis.

Acylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed that Bacillus subtilis and Bacillus anthracis encode the GCN5-related N-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of B. subtilis SatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of the B. anthracis Ames SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations into satA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 of BaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCE This work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.

Function and expression of N-acetyltransferases 1 and 2 are altered in lymphocytes in type 2 diabetes and obesity.

The cytosolic enzymes N-Acetyl Transferases 1 and 2 (NATs) transfer an acetyl group from acetyl-CoA to a xenobiotic substrate. NATs are regulated at the genetic and epigenetic levels by deacetylase enzymes such as sirtuins. The enzymatic expression of NAT1, NAT2, and SIRT1 was evaluated by flow cytometry, as well as the enzymatic activity of NATs by cell culture and HPLC analysis. Six SNPs were determined through genotyping. T2D patients (n = 29) and healthy subjects (n = 25) with a median age of 57 and 50, respectively, were recruited. An increased enzyme expression and a diminished NAT2 enzymatic activity were found in cells of T2D patients compared to the control group, while NAT1 was negatively correlated with body fat percentage and BMI. In contrast, Sirtuin inhibition increased NAT2 activity, while Sirtuin agonism decreased its activity in both groups. The analysis of NAT2 SNPs showed a higher frequency of rapid acetylation haplotypes in T2D patients compared to the control group, possibly associated as a risk factor for diabetes. The enzymatic expression of CD3+NAT2+ cells was higher in the rapid acetylators group compared to the slow acetylators group. The levels and activity of NAT1 were associated with total cholesterol and triglycerides. Meanwhile, CD3+NAT2+ cells and NAT2 activity levels were associated with HbA1c and glucose levels. The results indicate that NAT2 could be involved in metabolic processes related to the development of T2D, due to its association with glucose levels, HbA1c, and the altered SIRT-NAT axis. NAT1 may be involved with dyslipidaemias in people who are overweight or obese.

NatB Protects Procaspase-8 from UBR4-Mediated Degradation and Is Required for Full Induction of the Extrinsic Apoptosis Pathway.

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB (Naa20-/-) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20-/- MEFs exhibited reduced responsiveness to an extrinsic pro-apoptotic stimulus, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway appears to impact apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.

Altered Arylamine N-acetyltransferase 1 and miR-1290 Levels in Childhood Acute Lymphoblastic Leukemia: A Pilot Study.

BACKGROUND/AIM: Arylamine N-acetyltransferase 1 and 2 (NAT1 and NAT2) are drug-metabolizing enzymes that play a key role in the development of acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: This study evaluated NAT1 and NAT2 mRNA and protein expression and their enzymatic activity in peripheral blood mononuclear cells (PBMC) from patients with ALL (n=20) and healthy children (n=19) and explored the mechanisms that regulate these enzymes in ALL such as microRNAs (miR-1290, miR-26b) and SNPs. RESULTS: PBMC from patients with ALL showed a decrease in NAT1 mRNA and protein expression. In addition, NAT1 enzymatic activity was decreased in patients with ALL. There was no influence of SNP 559 C>T or 560 G>A on low NAT1 activity. The lower expression of NAT1 might be related to the loss of acetylated histone H3K14 in the NAT1 gene promoter in patients with ALL and the higher relative expression of miR-1290 in the plasma of patients with relapsed ALL compared with healthy controls. There were significantly fewer CD3+/NAT1+ double-positive cells in patients who relapsed compared with control subjects. Based on a t-distributed stochastic neighbor embedding algorithm, CD19+ cells that reappeared in patients with relapse showed low NAT1 expression. In contrast, for NAT2, there were no significant results. CONCLUSION: The expression and function of NAT1 and miR-1290 levels could be involved in modulating immune cells altered in ALL.

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 ß0 strand and instead contains a C-terminal ß7 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.

The role of arylamine N-acetyltransferases in chronic degenerative diseases: Their possible function in the immune system.

Since their discovery, arylamine N-acetyltransferases 1 and 2 (NAT1 and NAT2, respectively) have been associated with the metabolism of xenobiotics. NAT2 is the main factor in the therapeutic success of tuberculosis treatment due to its role in the biotransformation of isoniazid. However, researchers have started to investigate the possible participation of NAT1 and NAT2 (NATs) in carcinogenesis, although the mechanisms have not been elucidated fully. NATs enzymatic activity is essential in some types of cancer, such as breast cancer and acute lymphoblastic leukemia. Whether NAT1 and/or NAT2 participate in insulin resistance level in diabetes mellitus or in the immune system remains to be explored. Therefore, it is clear that its role in cell physiology has more implications than just metabolizing compounds.

Arabidopsis N-acetyltransferase activity 2 preferentially acetylates 1,3-diaminopropane and thialysine.

Polyamine acetylation has an important regulatory role in polyamine metabolism. It is catalysed by GCN5-related N-acetyltransferases, which transfer acetyl groups from acetyl-coenzyme A to the primary amino groups of spermidine, spermine (Spm), or other polyamines and diamines, as was shown for the human Spermidine/Spermine N1-acetyltransferase 1 (HsSSAT1). SSAT homologues specific for thialysine, a cysteine-derived lysine analogue, were also identified (e.g., HsSSAT2). Two HsSSAT1 homologues are present in Arabidopsis, namely N-acetyltransferase activity (AtNATA) 1 and 2. AtNATA1 was previously shown to be specific for 1,3-diaminopropane, ornithine, putrescine and thialysine, rather than Spm and spermidine. In the present study, in an attempt to find a plant Spm-specific SSAT, AtNATA2 was expressed in a heterologous bacterial system and catalytic properties of the recombinant protein were determined. Data indicate that recombinant AtNATA2 preferentially acetylates 1,3-diaminopropane and thialysine, throwing further light on AtNATA1 substrate specificity. Structural analyses evidenced that the preference of AtNATA1, AtNATA2 and HsSSAT2 for short amine substrates can be ascribed to different main-chain conformation or substitution of HsSSAT1 residues interacting with Spm distal regions. Moreover, gene expression studies evidenced that AtNATA1 gene, but not AtNATA2, is up-regulated by cytokinins, thermospermine and Spm, suggesting the existence of a link between AtNATAs and N1-acetyl-Spm metabolism. This study provides insights into polyamine metabolism and structural determinants of substrate specificity of non Spm-specific SSAT homologues.

Crystal structure of the phage-encoded N-acetyltransferase in complex with acetyl-CoA, revealing a novel dimeric arrangement.

Bacteriophages employ diverse mechanisms to facilitate the proliferation of bacteriophages. The Salmonella-infecting phage SPN3US contains a putative N-acetyltransferase, which is widely found in bacteriophages. However, due to low sequence similarity to the N-acetyltransferases from bacteria and eukaryotic cells, the structure and function of phage-encoded acetyltransferases are mainly unknown. This study determines the crystal structure of the putative N-acetyltransferase of SPN3US in complex with acetyl-CoA. The crystal structure showed a novel homodimeric arrangement stabilized by exchanging the C-terminal α-helix within the dimer. The following biochemical analyses suggested that the phage-encoded acetyltransferase might have a very narrow substrate specificity. Further studies are required to reveal the biochemical activity, which would help elucidate the interaction between the phage and host bacteria in controlling pathogenic bacteria.

Biochemical Characterization of Arylamine N-acetyltransferases From Vibrio vulnificus.

Vibrio vulnificus is a zoonotic bacterium that is capable of causing highly lethal diseases in humans; this pathogen is responsible for 95% of all seafood-related deaths in the United States. Arylamine N-acetyltransferases (NAT, E.C. 2.3.1.5) is a major family of xenobiotic-metabolizing enzymes that can biotransform aromatic amine chemicals. In this research, to evaluate the effect of NAT on acetyl group transformation in arylamine antibiotics, we first used sequence alignment to study the structure of V. vulnificus NAT [(VIBVN)NAT]. The nat gene encodes a protein of 260 amino acids, which has an approximate molecular mass of 30 kDa. Then we purified recombinant (VIBVN)NAT and determined the enzyme activity by PNPA and DTNB methods. The DTNB method indicates that this prokaryotic NAT has a particular substrate specificity towards aromatic substrates. However, (VIBVN)NAT lost most of its activity after treatment with high concentrations of urea and H2O2. In addition, we also explored the stability of the enzyme at different temperatures and pH values. In analyzing the influence of metal ions, the enzyme activity was significantly inhibited by Zn2+ and Cu2+. The kinetic parameters K m and V max were determined using hydralazine, isoniazid, 4-amino salicylic acid, and 4-chloro-3-methylaniline as substrates, and the T m , T agg and size distribution of (VIBVN)NAT were observed. In particular, a molecular docking study on the structure of (VIBVN)NAT was conducted to understand its biochemical traits. These results showed that (VIBVN)NAT could acetylate various aromatic amine substrates and contribute to arylamine antibiotic resistance in V. vulnificus.

Primary aromatic amines and cancer: Novel mechanistic insights using 4-aminobiphenyl as a model carcinogen.

Aromatic amines are an important class of human carcinogens found ubiquitously in our environment. It is estimated that 1 in 8 of all known or suspected human carcinogens is or can be converted into an aromatic amine, making the elucidation of their mechanisms of toxicity a top public health priority. Decades of research into aromatic amine carcinogenesis revealed a complex bioactivation process where Phase I and Phase II drug metabolizing enzymes catalyze N-oxidation and subsequent conjugation reactions generating the highly electrophilic nitrenium intermediate that reacts with and forms adducts on cellular macromolecules. Although aromatic amine-DNA adducts were believed to be the main driver of cancer formation, several studies have reported a lack of correlation between levels of DNA adducts and tumors. Using genetically modified mouse models, our laboratory and others observed several instances where levels of conventionally measured DNA adducts failed to correlate with liver tumor incidence following exposure to the model aromatic amine procarcinogen 4-aminobiphenyl. In this review we first provide a historical overview of the studies that led to a proposed mechanism of carcinogenesis caused by aromatic amines, where their bioactivation to form DNA adducts represents the central driver of this process. We then highlight recent mechanistic studies using 4-aminobiphenyl that are inconsistent with this mechanism which suggest novel drivers of aromatic amine carcinogenesis.

Humans and Chimpanzees Display Opposite Patterns of Diversity in Arylamine N-Acetyltransferase Genes.

Among the many genes involved in the metabolism of therapeutic drugs, human arylamine N-acetyltransferases (NATs) genes have been extensively studied, due to their medical importance both in pharmacogenetics and disease epidemiology. One member of this small gene family, NAT2, is established as the locus of the classic human acetylation polymorphism in drug metabolism. Current hypotheses hold that selective processes favoring haplotypes conferring lower NAT2 activity have been operating in modern humans' recent history as an adaptation to local chemical and dietary environments. To shed new light on such hypotheses, we investigated the genetic diversity of the three members of the NAT gene family in seven hominid species, including modern humans, Neanderthals and Denisovans. Little polymorphism sharing was found among hominids, yet all species displayed high NAT diversity, but distributed in an opposite fashion in chimpanzees and bonobos (Pan genus) compared to modern humans, with higher diversity in Pan species at NAT1 and lower at NAT2, while the reverse is observed in humans. This pattern was also reflected in the results returned by selective neutrality tests, which suggest, in agreement with the predicted functional impact of mutations detected in non-human primates, stronger directional selection, presumably purifying selection, at NAT1 in modern humans, and at NAT2 in chimpanzees. Overall, the results point to the evolution of divergent functions of these highly homologous genes in the different primate species, possibly related to their specific chemical/dietary environment (exposome) and we hypothesize that this is likely linked to the emergence of controlled fire use in the human lineage.

Butyrate interacts with benzo[a]pyrene to alter expression and activities of xenobiotic metabolizing enzymes involved in metabolism of carcinogens within colon epithelial cell models.

Butyrate helps to maintain colon homeostasis and exhibits chemopreventive effects in colon epithelium. We examined the interactive effects of butyrate and benzo[a]pyrene (BaP), dietary carcinogen, in regulation of expression of a panel of phase I and II xenobiotic metabolizing enzymes (XMEs) in human colon cells. In human colon carcinoma HCT-116 and HT-29 cell lines, butyrate alone increased mRNA levels of some enzymes, such as N-acetyltransferases (in particular NAT2). In combination with BaP, butyrate potentiated induction of cytochrome P450 family 1 enzymes (CYP1A1), aldo-keto reductases (AKR1C1) or UDP-glucuronosyltransferases (UGT1A1). There were some notable differences between cell lines, as butyrate potentiated induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) and UGT1A4 only in HCT-116 cells, and it even repressed AKR1C3 induction in HT-29 cells. Butyrate also promoted induction of CYP1, NQO1, NAT2, UGT1A1 or UGT1A4 in human colon Caco-2 cells, in a differentiation-dependent manner. Differentiated Caco-2 cells exhibited a higher inducibility of selected XME genes than undifferentiated cells. Butyrate increased induction of enzymatic activities of NATs, NQO1 and UGTs by BaP in HCT-116 and HT29 cells, whereas in differentiated Caco-2 cells it helped to increase only enzymatic activity of NQO1 and UGTs. Together, the present data suggest that butyrate may modulate expression/activities of several enzymes involved in metabolism of carcinogens in colon. In some cases (NAT2, UGT1 A1), this was linked to inhibition of histone deacetylases (HDAC), as confirmed by using HDAC inhibitor trichostatin A. These results may have implications for our understanding of the role of butyrate in regulation of XMEs and carcinogen metabolism in colon.

Genetic polymorphisms of arylamine N-acetyltransferases 1 and 2 and the likelihood of developing pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is one of the main causes of death in children and is associated with both genetic susceptibility and environmental factors. Genes encoding the arylamine N-acetyltransferases 1 and 2 (NAT1 and NAT2) isoenzymes are highly polymorphic among populations. Single-nucleotide polymorphism analysis was performed by real-time polymerase chain reaction from the genomic DNA of 225 healthy subjects and 57 children with ALL diagnoses. Significant associations were found between the development of ALL and the presence of the haplotypes NAT1*3 (Odds ratio [OR], 2.1), NAT1*4 (OR, 1.92), NAT2*6B (OR, 3.30), NAT2*6J (OR, 3.25) and NAT2*7A (OR, 2.45) and the NAT1 rapid (OR, 6.69) and NAT2 slow phenotypes (OR, 2.95). Our results indicate that haplotypes that provide rapid NAT1 and slow NAT2 acetylating phenotypes may influence the development of ALL in children.

Small-Molecule Acetylation Controls the Degradation of Benzoate and Photosynthesis in Rhodopseudomonas palustris.

The degradation of lignin-derived aromatic compounds such as benzoate has been extensively studied in Rhodopseudomonas palustris, and the chemistry underpinning the conversion of benzoate to acetyl coenzyme A (acetyl-CoA) is well understood. Here we characterize the last unknown gene, badL, of the bad (benzoic acid degradation) cluster. BadL function is required for growth under photoheterotrophic conditions with benzoate as the organic carbon source (i.e., light plus anoxia). On the basis of bioinformatics and in vivo and in vitro data, we show that BadL, a Gcn5-related N-acetyltransferase (GNAT) (PF00583), acetylates aminobenzoates to yield acetamidobenzoates. The latter relieved repression of the badDEFGAB operon by binding to BadM, triggering the synthesis of enzymes that activate and dearomatize the benzene ring. We also show that acetamidobenzoates are required for the expression of genes encoding the photosynthetic reaction center light-harvesting complexes through a BadM-independent mechanism. The effect of acetamidobenzoates on pigment synthesis is new and different than their effect on the catabolism of benzoate.IMPORTANCE This work shows that the BadL protein of Rhodopseudomonas palustris has N-acetyltransferase activity and that this activity is required for the catabolism of benzoate under photosynthetic conditions in this bacterium. R. palustris occupies lignin-rich habitats, making its benzoate-degrading capability critical for the recycling of this important, energy-rich biopolymer. This work identifies the product of the BadL enzyme as acetamidobenzoates, which were needed to derepress genes encoding benzoate-degrading enzymes and proteins of the photosynthetic apparatus responsible for the generation of the proton motive force under anoxia in the presence of light. In short, acetamidobenzoates potentially coordinate the use of benzoate as a source of reducing power and carbon with the generation of a light-driven proton motive force that fuels ATP synthesis, motility, transport, and many other processes in the metabolically versatile bacterium R. palustris.

Proteome-Wide Analysis of Trypanosoma cruzi Exponential and Stationary Growth Phases Reveals a Subcellular Compartment-Specific Regulation.

Trypanosoma cruzi, the etiologic agent of Chagas disease, cycles through different life stages characterized by defined molecular traits associated with the proliferative or differentiation state. In particular, T. cruzi epimastigotes are the replicative forms that colonize the intestine of the Triatomine insect vector before entering the stationary phase that is crucial for differentiation into metacyclic trypomastigotes, which are the infective forms of mammalian hosts. The transition from proliferative exponential phase to quiescent stationary phase represents an important step that recapitulates the early molecular events of metacyclogenesis, opening new possibilities for understanding this process. In this study, we report a quantitative shotgun proteomic analysis of the T. cruzi epimastigote in the exponential and stationary growth phases. More than 3000 proteins were detected and quantified, highlighting the regulation of proteins involved in different subcellular compartments. Ribosomal proteins were upregulated in the exponential phase, supporting the higher replication rate of this growth phase. Autophagy-related proteins were upregulated in the stationary growth phase, indicating the onset of the metacyclogenesis process. Moreover, this study reports the regulation of N-terminally acetylated proteins during growth phase transitioning, adding a new layer of regulation to this process. Taken together, this study reports a proteome-wide rewiring during T. cruzi transit from the replicative exponential phase to the stationary growth phase, which is the preparatory phase for differentiation.

In Salmonella enterica, OatA (Formerly YjgM) Uses O-Acetyl-Serine and Acetyl-CoA to Synthesize N,O-Diacetylserine, Which Upregulates Cysteine Biosynthesis.

L-Cysteine biosynthesis has been extensively analyzed in Salmonella enterica. The cysteine regulon contains the genes whose protein products are necessary to convert sulfate to sulfide, which is eventually reacted with O-acetyl-serine (OAS) to generate cysteine. The LysR type regulator, CysB, is required for activation of the cysteine regulon, and its interaction with various cys genes has been thoroughly characterized. Results from previous studies by others, suggested that OAS undergoes a spontaneous O- to N- migration to produce N-acetyl-serine (NAS), and that NAS is the true signal sensed by CysB. It was unclear, however, whether such migration occurred spontaneously in vivo or if NAS was generated enzymatically. Work reported herein characterizes a S. enterica N-acetyltransferase, OatA (formerly YjgM), which acetylates the N α-amino group of OAS, producing N,O-diacetyl-serine (DAS) at the expense of acetyl-CoA. We isolated OatA to homogeneity and performed its initial biochemical characterization. The product of the OatA reaction was isolated by HPLC and confirmed by mass spectrometry to be DAS; OatA did not acetylate NAS, consistent with the conclusion that OatA is an N-acetyltransferase, not an O-acetyltransferase. Binding of OAS to OatA appears to be positively cooperative with the apparent K0.5 for OAS determined to be 0.74 mM, the kcat was 1.05 s-1, and the catalytic efficiency of the enzyme (k cat/K0.5 ) was 1.4 × 103 M-1 s-1. Size exclusion chromatography indicated that OatA was a monomer in solution. In S. enterica, overexpression of oatA led to shorter lag times on sulfate-limiting medium and that these delayed lag times were due to increased expression of the cysteine regulon, as indicated by RT-qPCR results. OatA is the first Gcn5-related N-acetyltransferase (aka GNAT) involved in the regulation of amino acid biosynthetic genes in Salmonella. On the basis of results of transcriptomics studies performed by other investigators, we hypothesize that DAS may play a role in biofilm formation in S. enterica and other bacteria.