Arylamine N-acetyltransferase in Balb/c mice: identification of a novel mouse isoenzyme by cloning and expression in vitro.
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ABSTRACT:
SUBMITTER: Kelly SL
PROVIDER: S-EPMC1137235 | biostudies-other | 1994 Sep
REPOSITORIES: biostudies-other
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Project description:New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules-triazoles-in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.
| S-EPMC4875111 | biostudies-literature
Project description:A single-nucleotide polymorphism in the human arylamine N-acetyltransferase 2 (Nat2) gene has recently been identified as associated with insulin resistance in humans. To understand the cellular and molecular mechanisms by which alterations in Nat2 activity might cause insulin resistance, we examined murine ortholog Nat1 knockout (KO) mice. Nat1 KO mice manifested whole-body insulin resistance, which could be attributed to reduced muscle, liver, and adipose tissue insulin sensitivity. Hepatic and muscle insulin resistance were associated with marked increases in both liver and muscle triglyceride (TAG) and diacylglycerol (DAG) content, which was associated with increased PKC? activation in liver and increased PKC? activation in skeletal muscle. Nat1 KO mice also displayed reduced whole-body energy expenditure and reduced mitochondrial oxygen consumption in white adipose tissue, brown adipose tissue, and hepatocytes. Taken together, these studies demonstrate that Nat1 deletion promotes reduced mitochondrial activity and is associated with ectopic lipid-induced insulin resistance. These results provide a potential genetic link among mitochondrial dysfunction with increased ectopic lipid deposition, insulin resistance, and type 2 diabetes.
| S-EPMC5748223 | biostudies-literature
Project description:N-alpha-terminal acetylation is a modification process that occurs cotranslationally on most eukaryotic proteins. The major enzyme responsible for this process, N-alpha-terminal acetyltransferase, is composed of the catalytic subunit ARD1A and the auxiliary subunit NAT1. We cloned, characterized, and studied the expression pattern of Ard1b (also known as Ard2), a novel homolog of the mouse Ard1a. Comparison of the genomic structures suggests that the autosomal Ard1b is a retroposed copy of the X-linked Ard1a. Expression analyses demonstrated a testis predominance of Ard1b. A reciprocal expression pattern between Ard1a and Ard1b is also observed during spermatogenesis, suggesting that Ard1b is expressed to compensate for the loss of Ard1a starting from meiosis. Both ARD1A and ARD1B can interact with NAT1 to constitute a functional N-alpha-terminal acetyltransferase in vitro. The expression of ARD1B protein can be detected in mouse testes but is delayed until the first appearance of round spermatids. In a cell culture model, the inclusion of the long 3' untranslated region of Ard1b leads to reduction of luciferase reporter activity, which implicates its role in translational repression of Ard1b during spermatogenesis. Our results suggest that ARD1B may have an important role in the later course of the spermatogenic process.
| S-EPMC2849813 | biostudies-literature
Project description:The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut.To determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards "(MOUSE)NAT2-specific" arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated.Comparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes.Three non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours.
| S-EPMC4258814 | biostudies-literature
Project description:Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned from Mycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli and M. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT from M. smegmatis and cross-reacts with recombinant NAT from M. tuberculosis. Overexpression of mycobacterial nat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatis as the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.
| S-EPMC93516 | biostudies-literature
Project description:Arylamine N-acetyltransferases (NATs) are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme.We have determined that NAT from Pseudomonas aeruginosa, which has been described as a model for NAT from M. tuberculosis, follows a Ping Pong Bi Bi kinetic mechanism. We also describe substrate inhibition by 5-aminosalicylic acid, in which the substrate binds both to the free form of the enzyme and the acetyl coenzyme A-enzyme complex in non-productive reaction pathways. The true kinetic parameters for the NAT-catalysed acetylation of 5-aminosalicylic acid with acetyl coenzyme A as the co-factor have been established, validating earlier approximations.This is the first reported study investigating the kinetic mechanism of a bacterial NAT enzyme. Additionally, the methods used herein can be applied to investigations of the interactions of NAT enzymes with new chemical entities which are NAT ligands. This is likely to be useful in the design of novel potential anti-tubercular agents.
| S-EPMC1851014 | biostudies-literature
Project description:Background:Human arylamine N-acetyltransferase 2 (NAT2) gene has a key role in xenobiotic metabolism through the conjugation of acetyl group to xenobiotic substances. NAT2 has been suggested as a susceptibility factor in endometriosis; however, the results of studies have been controversial. In this study, the association of NAT2 polymorphisms with susceptibility to endometriosis was evaluated in an Iranian population. Methods:This is an association study and totally 141 women with diagnosis of endometriosis and 158 healthy women as control group were analyzed for NAT2 gene polymorphisms (C481T, A803G, G857A and G590A) by PCR-RFLP methods. Results:The 590 GA genotype was significantly lower (p=0.001; OR=0.42, 95% CI: 0.25-0.71) in the patients (38.3%) than the control group (55.1%). The 590A allele was significantly lower (p=0.033; OR=0.69, 95% CI: 0.49-0.79) in the patients (31.2%) compared with the controls (39.6%). Analysis of haplotypes showed that NAT2 481C, 803A, 590A, 587A combination was significantly different between the case and control women (p= 0.029; OR=3.11, 95% CI: 1.13-8.52). Conclusion:The NAT2 G590A SNP may be associated with susceptibility to endometriosis and the 590A allele may have a protective role in development of endometriosis. The NAT2 481C, 803A, 590A, 587A haplotype was associated with a higher risk of endometriosis in Iranian population.
| S-EPMC6064003 | biostudies-literature
Project description:Arylamine N-acetyltransferases (NATs) detoxify arylamines and hydrazine xenobiotics by catalyzing their N-acetylation, which prevents their bioactivation. Here, we reveal how structural dynamics impact NAT protein function. Our data suggest that there are multiple conformations in the catalytic cavity of hamster NAT2 that exchange on the millisecond time scale and enable NATs to accommodate substrates of varying size. The regions spanning N177-L180 and D285-F288, which form unique structures in mammalian NATs, possess inherent motions on the nanosecond time scale. The latter segment becomes more restricted in its motions upon substrate binding according to our NMR XNOE data. This greater rigidity appears to stem from interactions with the substrate. Finally, NAT acetylation has been suggested to protect these enzymes from ubiquitination. Our NMR data on a catalytically active state of hamster NAT2 suggest that structural rearrangements caused by its acetylation might contribute to this protection.
| S-EPMC2748875 | biostudies-literature
Project description:Mapping proteins at a specific subcellular location is essential to gaining detailed insight on local protein dynamics. We have developed an enzymatic strategy to label proteins on a subcellular level using arylamine N-acetyltransferase (NAT). The NAT enzyme activates an arylhydroxamic acid functionality into a nitrenium ion that reacts fast, covalently, and under neutral conditions with nucleophilic residues of neighboring proteins. The electron density on the aromatic ring proved important for probe activation as strong labeling was only observed with an arylhydroxamic acid bearing an electron donating substituent. We further demonstrate that, using this electron rich arylhydroxamic acid, clear labeling was achieved on a subcellular level in living cells that were transfected with a genetically targeted NAT to the nucleus or the cytosol.
| S-EPMC6143282 | biostudies-literature
Project description: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.
| S-EPMC7497018 | biostudies-literature