Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding

Background: 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. Methods: 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. Results: 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. Conclusions: 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. </p

From arylamine N-acetyltransferase to folate-dependent acetyl CoA hydrolase : impact of folic acid on the activity of (HUMAN)NAT1 and its homologue (MOUSE)NAT2

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer

Investigating the endogenous role of human n-acetyltransferase 1, as potential breast cancer biomarker, using chemical biology

Human N-acetyltransferase 1 (hNAT1) is one of the ten most highly overexpressed genes in oestrogen-receptor-positive (ER+ve) breast cancers and its overexpression is strongly related to tumour grade. N-acetyltransferases from prokaryotic and eukaryotic kingdoms catalyse the transfer of an acetyl group from acetyl coenzyme A (CoA) to a variety of arylamines and arylhydrazines. While the other human isoenzyme hNAT2 has widely been assessed as a phase-II xenobiotic metabolising enzyme, the exact endogenous role of hNAT1 is still unknown. The association of hNAT1 with ER levels in breast tumours may imply a role in cancer progression, making it an attractive potential biomarker for ER+ve breast cancers and/or a novel therapeutic target. Mice offer a good animal model for investigations on human NATs: hNAT1 and mouse NAT2 (mNat2) are orthologous genes and the corresponding proteins, hNAT1 and mNat2, are homologous on the basis of sequence identity (82&percent;), substrate specificity and expression profile. Investigating selective inhibitors for hNAT1 and mNat2 is described in this thesis with the aim of using these inhibitors to aid in determining the in vivo function of hNAT1 and its mouse homologue. Naphthoquinone 1 was identified as a selective competitive inhibitor for hNAT1 and mNat2 (IC50,hNAT1=1.65 μM and IC50,mNat2=1.86μM) from a high-throughput screening of 5000 drug-like compounds against five distinct pure recombinant NATs. This compound also displays a distinctive colour change from red (λmax = 484nm) to blue (λmax = 610nm) in the presence of both hNAT1 and mNat2, but not the other human and murine isoenzymes. The colourimetric change was also observed by titration of compound 1 with an alkali. Physicochemical, biochemical and computational studies were conducted on naphthoquinone 7, an analogue of 1 with improved pharmacological properties (IC50,mNat2=0.99μM) and colour intensity, to support the hypothesis that the colour change event is related to deprotonation of the sulfonamide-NH of the ligand by the side-chain guanidine of Arg127 within the active site of both enzymes, hNAT1 and mNat2. Furthermore, the comparison of the arylamine substrate profiles of eight different mammalian NATs, alongside their preferences for inhibitor 7, provided substantial elements on the key role of Phe125, Arg127 and Tyr129 on isoenzyme selectivity for both substrate and inhibitor. This supports the development of this family of naphthoquinones as highly selective inhibitors of hNAT1 and mNat2 to elucidate the endogenous role of these proteins via Chemical Genetics, and as colourimetric biosensors to detect and quantify hNAT1 in breast cancer tissues. Selective recognition of hNAT1 by antibody allowed a preliminary estimation of the enzyme overex-pressed in the breast cancer cell line ZR-75-1: 1pg per cell, for which the binding affinity and the colourimetric properties of compound 7 were found to need further improvement. With the goal of improving both inhibitory potency and colourimetric properties of compound 7, a set of analogues varying at R1, R2 and R3 positions was chemically synthesised. The resulting substitutions al-tered inhibitory activity, range of colour change, molar extinction coefficient and acidity of the naphthoquinone derivatives, with compound 20 offering a tenfold increase in inhibitory potency towards both hNAT1 and mNat2 over 7, but less suitable colourimetric properties. Besides investigating the ability of different eukaryotic and prokaryotic NATs to use also n-propionylCoA as substrate, hNAT1 and mNat2 were exclusively identified to act as folate-dependent acetylCoA hydrolases compared to a panel of diverse eukaryotic and prokaryotic NATs, from which new hypotheses are proposed in the endogenous role of these enzymes in relation to folate, fat catabolism and cancer

A Door-to-Door Waste Collection System Case Study: A Survey on its Sustainability and Effectiveness

Municipal waste management is a relevant topic these days, in its relation to sustainable and environmental concerns. Sorting waste fractions at home for a door-to-door collection system proves to positively affect the environmental impacts of waste management strategies both by reducing the amounts of the waste landfilled and by originating new circular economies. However, the environmental impact caused by both waste collection and transport, together with waste quality, should be carefully evaluated to assess the sustainability of such a collection system. In order to evaluate the logistic and environmental effectiveness of a newly implemented door-to-door collection system in Altamura, a mid-sized town in Southern Italy, a survey was designed and submitted to a sample of citizens. The results obtained from the 385 completed surveys show that the door-to-door collection of glass waste is inefficient since most of the designated bins remain partially filled and less frequently delivered; citizens are more motivated to adequately collect sorted waste fractions upon receiving information about the subsequent environmental benefits and outcomes of the fractions collected; a high percentage of people still use disposable items in their daily life. Possible changes to the weekly bins collection schedule have been proposed in order to have a more proficient and environmentally sustainable waste collection service in the town. The survey is part of a project aiming at developing a smart device to support users in home waste management

Arylamine N-acetyltransferases - from drug metabolism and pharmacogenetics to identification of novel targets for pharmacological intervention

Arylamine N-acetyltransferases (NATs) are defined as xenobiotic metabolizing enzymes, adding an acetyl group from acetyl coenzyme A (CoA) to arylamines and arylhydrazines. NATs are found in organisms from bacteria and fungi to vertebrates. Several isoenzymes, often polymorphic, may be present in one organism. There are two functional polymorphic NATs in humans and polymorphisms in NAT2 underpinned pharmacogenetics as a discipline. NAT enzymes have had a role in important metabolic concepts: the identification of acetyl-CoA and endogenous metabolic roles in bacteria and in eukaryotic folate metabolism. In fungi, NAT is linked to formation of unique metabolites. A broad and exciting canvas of investigations has emerged over the past five years from fundamental studies on NAT enzymes. The role of human NAT1 in breast cancer where it is a biomarker and possible therapeutic target may also underlie NAT's early appearance during mammalian fetal development. Studies of NAT in Mycobacterium tuberculosis have identified potential therapeutic targets for tuberculosis whilst the role of NATs in fungi opens up potential toxicological intervention in agriculture. These developments are possible through the combination of genomics, enzymology and structural data. Strong binding of CoA to Bacillis anthracis NAT may point to divergent roles of NATs amongst organisms as does differential control of mammalian NAT gene expression. The powerful combination of phenotypic investigation following genetic manipulation of NAT genes from mice to mycobacteria has been coupled with generation of isoenzyme-specific inhibitors. This battery of molecular and systems biology approaches heralds a new era for NAT research in pharmacology and toxicology

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water. In the gut flora, they activate azo pro-drugs, which are used for the treatment of inflammatory bowerl disease, releasing the active component 5-aminosalycilic acid. The bacterium P. aeruginosa,/em> has three azoreductase genes, paAzoR1, paAzoR2 and paAzoR3, which as recombinant enzymes have been shown to have different substrate specificities. The mechanisms of azoreduction relies upon tautomerisation of the substrate to the hydrazone form. We report here the characterization of the P. aeruginosa azoreductase enzymes, including determining their thermostability, cofactor preference and kinetic constants against a range of their favoured substrates. The expression levels of these enzymes during growth of P. aeruginosa are altered by the presence of azo substrates. It is shown that enzymes that were originally described as azoreductases, are likely to act as NADH quinone oxidoreductases. The low sequence identities observed among NAD(P)H quinone oxidoreductase and azoreductase enzymes suggests convergent evolution.The full-text of this article is not currently available in ORA, but the original publication is available at springerlink.com (which you may be able to access via the publisher copy link on this record page). Citation: Ryan, A. et al. (2010). 'Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases', Protein & Cell 1(8), 780-790

Temperature stability of proteins essential for the intracellular survival of Mycobacterium tuberculosis

In Mycobacterium tuberculosis, the genes hsaD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) and nat (arylamine N-acetyltransferase) are essential for survival inside of host macrophages. These genes act as an operon and have been suggested to be involved in cholesterol metabolism. However, the role of NAT in this catabolic pathway has not been determined. In an effort to better understand the function of these proteins, we have expressed, purified and characterized TBNAT (NAT from M. tuberculosis) and HsaD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) from M. tuberculosis. Both proteins demonstrated remarkable heat stability with TBNAT and HsaD retaining >95% of their activity after incubation at 60 degrees C for 30 min. The first and second domains of TBNAT were demonstrated to be very important to the heat stability of the protein, as the transfer of these domains caused a dramatic reduction in the heat stability. The specific activity of TBNAT was tested against a broad range of acyl-CoA cofactors using hydralazine as a substrate. TBNAT was found to be able to utilize not just acetyl-CoA, but also n-propionyl-CoA and acetoacetyl-CoA, although at a lower rate. As propionyl-CoA is a product of cholesterol catabolism, we propose that NAT could have a role in the utilization of this important cofactor