Density Functional Theory Study on the Histidine-assisted Mechanism of Arylamine N-Acetyltransferase Acetylation

Arylamine N-acetyltransferases （NATs, EC 2.3.1.5） catalyze the N-acetylation of primary arylamines, and play a key role in the biotransformation and metabolism of drugs, carcinogens, etc. In this paper, three possible reaction mechanisms are investigated and the results indicate that if the acetyl group directly transfers from the donor to the acceptor, the high activation energies will make it hard to obtain the target products. When using histidine to mediate the acetylation process, these energies will drop in the 15-45 kJ/mol range. If the histidine residue is protonated, the corresponding energies will be decreased by about 35-87 kJ/mol. The calculations predict an enzymatic acetylation mechanism that undergoes a thiolate-imidazolium pair, which agrees with the experimental results very well.

Arylamine N-acetyltransferases: a new inhibitor of apoptosis in HepG2 cells

Objective: To explore how arylamine N-acetyltransferases (NATs) is related to cell apoptosis. Methods: NAT activity in apoptotic HepG2 cells was measured using high performance liquid chromatography (HPLC); the apoptosis rate of HepG2 cells acted upon by an NAT inhibitor was measured using flow cytometry. Results: NAT activity was lowered in apoptotic HepG2 cells; apoptosis rate induced by camptothecin (CAM) increased after inhibition of NAT activity in HepG2 cells. Conclusion: NAT can inhibit apoptosis in HepG2 cells.

Inflammatory cytokines suppress arylamine N-acetyltransferase 1 in cholangiocarcinoma cells

瞄准：为了在芳基胺上评估煽动性的 cytokines 的效果， N-acetyltransferase 1 (NAT1 ) 它是 phase-II 酶在在食物，药和环境发现的芳香、杂环的胺的简历转变包含了。方法：人的 cholangiocarcinoma KKU-100 房间与专业版的混合物被对待为 48 h 的煽动性的 cytokines (interferon-gamma， interleukin-1beta 和肿瘤坏死 factor-alpha ) ，和在 NAT1 活动的效果被高效液相色谱法估计，当 NAT1 表示被反向抄写的聚合酶链反应决定时。房间上的氧化压力被氮的氧化物，超级氧化物阴离子和谷胱甘肽(GSH ) 的形成检验层次。房间也与 S-nitroso-glutathione (GSNO ) 被对待，一个氮的氧化物施主，与煽动性的 cytokines 获得了看回答是否类似于那些。结果：Cytokines 压制了 NAT1 活动，减少没有影响 Km 的 Vmax。Cytokines 也在氮的氧化物生产并且在减少谷胱甘肽(GSH ) 和 GSH 二硫化物的氧化还原作用比率的正式就职上有重要影响。没有影响 GSH 比率，有为 2-48 h 的 GSNO 的处理减少了 NAT1 活动。而且，煽动性的 cytokines 和 GSNO 压制了 NAT1 mRNA 表示。结论：这些调查结果显示在 NAT1 的发炎和抑制之间的一个协会，它也许贡献调停化学药品的毒性和致癌作用。

A pilot study of the modulation of sirtuins on arylamine N-acetyltransferase 1 and 2 enzymatic activity

Arylamine N-acetyltransferase(NAT; E.C. 2.3.1.5) enzymes are responsible for the biotransformation of several arylamine and hydrazine drugs by acetylation. In this process, the acetyl group transferred to the acceptor substrate produces NAT deacetylation and, in consequence, it is susceptible of degradation. Sirtuins are protein deacetylases, dependent on nicotine adenine dinucleotide,which perform post-translational modifications on cytosolic proteins. To explore possible sirtuin participation in the enzymatic activity of arylamine NATs, the expression levels of NAT1, NAT2,SIRT1 and SIRT6 in peripheral blood mononuclear cells(PBMC) from healthy subjects were examined by flow cytometry and Western blot. The in situ activity of the sirtuins on NAT enzymatic activity was analyzed by HPLC, in the presence or absence of an agonist(resveratrol) and inhibitor(nicotinamide) of sirtuins. We detected a higher percentage of positive cells for NAT2 in comparison with NAT1, and higher numbers of SIRT1t cells compared to SIRT6 in lymphocytes. In situ NAT2 activity in the presence of NAM inhibitors was higher than in the presence of its substrate, but not in the presence ofresveratrol. In contrast, the activity of NAT1 was not affected by sirtuins. These results showed that NAT2 activity might be modified by sirtuins.

Probing the architecture of the Mycobacterium marinum arylamine N-acetyltransferase active site

Treatment of latent tuberculosis infection remains an important goal of global TB eradication.To this end,targets that are essential for intracellular survival of Mycobacterium tuberculosis are particularly attractive.Arylamine N-acetyltransferase(NAT)represents such a target as it is,along with the enzymes encoded by the associated gene cluster,essential for mycobacterial survival inside macrophages and involved in cholesterol degradation.Cholesterol is likely to be the fuel for M.tuberculosis inside macrophages.Deleting the nat gene and inhibiting the NAT enzyme prevents survival of the microorganism in macrophages and induces cell wall alterations,rendering the mycobacterium sensitive to antibiotics to which it is normally resistant.To date,NAT from M.marinum(MMNAT)is considered the best available model for NAT from M.tuberculosis(TBNAT).The enzyme catalyses the acetylation and propionylation of arylamines and hydrazines.Hydralazine is a good acetyl and propionyl acceptor for both MMNAT and TBNAT.The MMNAT structure has been solved to 2.1Åresolution following crystallisation in the presence of hydralazine and is compared to available NAT structures.From the mode of ligand binding,features of the binding pocket can be identified,which point to a novel mechanism for the acetylation reaction that results in a 3-methyltriazolo[3,4-a]phthalazine ring compound as product.

What a Role did Histidine Residue Play in Arylamine N-Acetyltransferase 2 Acetylation？ A Quantum Chemistry Study

Arylamine N-acetyltransferases （NATs, EC 2.3.1.5） catalyze an acetyl group transfer from acetyl coenzyme A （AcCoA） to primary arylamines and play a very important role in the metabolism and bioactivation of drugs and carcinogens. Experiments revealed that His-107 was likely the residues responsible for mediating acetyl transfer. The full catalytic mechanism of acetylation process has been examined by density functional theory. The results indicate that, if the acetyl group is directly transferred from the donor, p-nitrophenyl acetate, to the acceptor, cysteine, the high activation energy will be a great hindrance. These energies have dropped in a little range of 20-25 kJ/mol when His-107 assisted the transfer process. However, when protonated His-107 mediated the reaction, the activation energies have been dropped about 73-85 kJ/mol. Our calculations strongly supported an enzyme acetylation mechanism that experiences a thiolate-imidazolium pair, and verified the presumption from experiments.

Identification of arylamine N-acetyltransferase inhibitors as an approach towards novel anti-tuberculars

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.

Arylamine N-acetyltransferase 2 genotype-dependent N-acetylation of isoniazid in cryopreserved human hepatocytes

Cryopreserved human hepatocytes were used to investigate the role of arylamine Nacetyltransferase 2(NAT2; EC 2.3.1.5) polymorphism on the N-acetylation of isoniazid(INH). NAT2 genotype was determined by Taqman allelic discrimination assay and INH N-acetylation was measured by high performance liquid chromatography. INH N-acetylation rates in vitro exhibited a robust and highly significant(P o0.005) NAT2 phenotype-dependent metabolism. N-acetylation rates in situ were INH concentration-and time-dependent. Following incubation for 24 h with 12.5 or 100 mmol/L INH, acetylINH concentrations varied significantly(P = 0.0023 and P = 0.0002) across cryopreserved human hepatocytes samples from rapid, intermediate, and slow acetylators, respectively. The clear association between NAT2 genotype and phenotype supports use of NAT2 genotype to guide INH dosing strategies in the treatment and prevention of tuberculosis.

CuCl-catalyzed Oxidative N-Demethylation of Arylamines with tButyl Hydroperoxide

CuCl-catalyzed oxidative N-demethylation of arylamines proceeded in the presence of tert-butyl hydroperoxide. The one-electron transfer route of oxidative N-demethylation competed favorably with the H-atom abstraction route.

Polymorphism of N-acetyltransferase 2 (NAT2) Gene Polymorphism in Shanghai population: Occupational and Non-occupational Bladder Cancer Patient Groups

Objective Arylamine N-acetyltransferases (NATs) are involved in the detoxification of aromatic amines and hydrazine. In order to explore the possible association of NAT2 polymorphism with bladder cancer risk in benzidine exposed or non-exposed Chinese individuals, healthy subjects, subjects with bladder cancer of a former benzidine exposed cohort in Shanghai dyestuff industry and a group of bladder cancer patients without known occupational exposure to aromatic amines were genotyped for NAT2 gene polymorphism. Methods NAT2 genotyping was performed with a set of RFLP procedures at seven major polymorphic loci of gene coding area: G191A, C282T, T341C, C481T, G590A, A803G and G857A. Results The wild allele NAT2 *4 was the most prevalent allele (59%) in healthy individuals. The alleles NAT2*6A and NAT2*7B were also frequently observed (21% and 17%, respectively). In contrast to Caucasians, the percentage of slow acetylators was lower (12% in Chinese vs. 58% in Caucasians, P<0.001). No relevant differences were observed for homogenous rapid, heterogeneous rapid/slow and homogeneous slow acetylation genotypes between the healthy subjects and both groups of bladder cancer patients. Conclusion The present work did not support the association of slow acetylating genotypes of NAT2 gene with elevated risk of bladder cancer in Chinese whereas it was documented as an important genetically determined risk factor in Caucasians. Different mechanisms might play a role in individual susceptibility to bladder cancer related with aromatic amine exposure in various races or ethnic groups.

Two Dehydroabietic Acid-based Arylamines: Synthesis, Crystal Structure and Fluorescent Properties

Two dehydroabietic acid-based arylamines have been synthesized and characterized by FTIR, 1H NMR, 13C NMR, MS spectra and elemental analysis. Their spatial structures were determined by X-ray diffraction analysis. UV-Vis absorption and fluorescence spectral characteristics of these compounds in methanol were investigated. Their fluorescence emission spectra in different polarity solvents were further evaluated. Fluorescent properties and structural relationship of the compounds showed that fluorescence intensity and quantum yield inversely increase with the non-coplanar degree. In addition, the solvent polarity has different effects on the fluorescence emission spectra of two compounds.

N-acetyltransferase 2: Slow, intermediate or fast? A booming question of the molecular epidemiology in cancer research

Throughout history, humanity has referred to reactions occurring with food, plants and, recently, medicines or drugs. The increase in pulmonary tuberculosis cases and the availability of treatment showed that genetic human differences can interfere in the capacity to metabolize drugs. There are remarkable genetic polymorphisms of N-acetyltransferase 2 (NAT2) activity that have been associated with different levels of susceptibility to developing many kinds of cancers. This review considers the field as an open window for the application of molecular epidemiology tools that led to the development of pharmacogenomics. We cover historical data and the most recent knowledge about NAT2 genetic polymorphisms and its distribution in different populations, which is an important concept being incorporated in epidemiological studies of cancer risk. We present up to date information about these studies, including meta-analysis based on the NAT2 distribution in different types of cancer. A critical broad at advances in NAT2 research, high-lighting recent studies related to NAT2 alleles in cancer susceptibility. Although there are multifactorial aspects involved in cancer risk, the variability in NAT2 allelic frequency can be related to carcinogenesis through alterations in the metabolic rate after exposure to carcinogens.

Night-time Rise in Rat Pineal N-Acetyltransferase due to Carbaryl Administration Is Reduced by Propranolol Treatment

The purpose of the present study was to examine the effects of administration of sublethal doses of carbaryl on nighttime rat pineal melatonin synthesis in the presence and absence of propranolol, a β-adrenergic receptor antagonist. Two groups of adult male albino rats were administered orally N-methyl-l-naphthylcarbamate (carbaryl) (8. 33mg/kg BW daily in corn oil) for six successive days; another two groups received corn oil only.On the last day of carbaryl treatment, half of the animals received an intraperitoneal injection of propranolol (20 mg/kg body weight, one hour before lights off). The other two groups were given a saline injection. Four hours after darkness onset, pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities as well as pineal concentrations of 5-hydroxytryptophan (5HTP), serotonin (5HT),5-hydroxyindole acetic acid (5HIAA) and pineal and serum melatonin levels were measured. Nocturnal NAT activity was increased due to carbaryl administration but the pesticide was ineffective in stimulating NAT activity in rats treated with propranolol.Pineal 5HT was decreased due to carbaryl administration but 5HTP and 5HIAA levels were unaffected. Pineal and serum melatonin levels were decreased due to propranolol treatment. The results indicate that carbaryl may influence pineal NAT activity by a mechanism that involves β-adrenergic neural transmission.

N-芳基烯胺酮衍生物的高效合成方法

以环氧乙烷衍生物和芳胺为原料,醋酸·三氟化硼/乙酰丙酮协同促进下、1,4-二氧六环溶剂中80℃经原位开环-芳基迁移过程,合成了一系列N-芳基烯胺酮衍生物,收率优良.核磁共振氢谱、红外光谱和高分辨率质谱证实了产物的结构,其中化合物3a的结构得到单晶衍射的确认.

Evolutionary genomics analysis reveals gene expansion and functional diversity of arylalkylamine N-acetyltransferases in the Culicinae subfamily of mosquitoes

Arylalkylamine N-acetyltransferase(aaNAT),considered a potential new insecticide target,catalyzes the acetylation of arylalkylamine substrates such as serotonin and dopamine and,hence,mediates diverse functions in insects.However,the origin of insect aaNATs(iaaNATs)and the evolutionary process that generates multiple aaNATs in mosquitoes remain largely unknown.Here,we have analyzed the genomes of 33 species to explore and expand our understanding of the molecular evolution of this gene family in detail.We show that aaNAT orthologs are present in Bacteria,Cephalochordata,Chondrichthyes,Cnidaria,Crustacea,Mammalia,Placozoa,and Teleoste,as well as those from a number of insects,but are absent in some species of Annelida,Echinozoa,and Mollusca as well as Arachnida.Particularly,more than 10 aaNATs were detected in the Culicinae subfamily of mosquitoes.Molecular evolutionary analysis of aaNAT/aaNAT-like genes in mosquitoes reveals that tandem duplication events led to gene expansion in the Culicinae subfamily of mosquitoes more than 190 million years ago.Further selection analysis demonstrates that mosquito aaNATs evolved under strongly positive pressures that generated functional diversity following gene duplication events.Overall,this study may provide novel insights into the molecular evolution of the aaNAT family in mosquitoes.

液-液相转移催化法合成羧基苯氧基乙酸衍生物的研究

用液-液相转移催化法合成了19个羰芳氧基苯氧基乙酸芳酯和19种羰芳胺基苯氧基乙酰芳胺,并初步考查这些化合物对小麦生长的促进作用。

中国黄海柄海鞘的化学成分

目的:对采自中国黄海的柄海鞘(Styela clava)的化学成分进行研究,从中寻找有生物活性的次生代谢产物。方法:用硅胶柱层析和凝胶柱层析对柄海鞘的丙酮提取物进行分离纯化,根据其化学性质,结合现代波谱技术(MS,NMR等),对得到的化合物进行结构鉴定。结果:分离得到3个类胡萝卜素和1个芳香胺类化合物,其结构分别鉴定为mytiloxanthinone(1)、fucoxanthin(2)、all-trans-astaxanthin(3)和naphthalen-2-yl-phenyl-amine(4)。结论:这些化合物均系首次从柄海鞘中分离得到,并首次对化合物mytiloxanthinon(1)和fucoxanthin(2)的1H和13C NMR数据进行了全归属。

有机钼酸酯与芳胺抗氧剂在合成润滑油中的抗氧协同作用

利用差示扫描量热计(DSC)评价了钼酸酯(MoON)和p,p-二异辛基二苯胺抗氧剂(DODPA)在聚α-烯烃合成润滑油中的热氧化安定性。在静态和动态DSC氧化实验中,MoON与DODPA复合后,均可有效提高基础油的起始氧化温度和氧化诱导时间。热油氧化实验结果也表明,MoON与DODPA复合后,能有效地抑制聚α-烯烃润滑油的粘度增加。因此,MoON与DODPA具有良好的抗氧协同作用。

一种芳胺低聚物抗氧剂的抗氧化性能

选用加压差示扫描量热法和烘箱氧化法考察了一种芳胺低聚物抗氧剂在基础油(酯)中、锂基润滑脂中的抗氧化效果,用氧化安定性和腐蚀性试验考察了该抗氧剂在AS5780航空涡轮发动机油中的性能。结果表明,芳胺低聚物抗氧剂在基础油(酯)中具有突出的抗氧化效果;在润滑脂和航空涡轮发动机油中,都具有优异的高温抗氧化性能。

催化氧化法处理含芳胺模拟废水的研究

文章以自制的Fe3O4纳米颗粒为催化剂,采用催化氧化法处理含苯胺和对硝基苯胺2种芳胺模拟废水。通过单因素条件实验分别考察了反应时间、反应温度、pH值、催化剂用量及H2O2的用量等因素的变化对模拟废水中芳胺去除率的影响。结果表明,处理100 mL含苯胺模拟废水的优化条件为:反应时间60 min,温度40℃,pH=3.5,H2O2(15%)的用量1 mL,催化剂Fe3O4用量11 mg;处理100 mL含对硝基苯胺废水优化条件为:反应时间4 h,温度60℃,pH=3.6,H2O2(15%)的用量1.2 mL,催化剂Fe3O4用量9 mg。在优化条件下的重复实验表明,2种含芳胺废水中芳胺的去除率均达99%。