RESUMO
Bipolar disorder is a major psychiatric disorder associated with cognitive impairment and a high suicide rate. Frontline therapy for this condition includes lithium (Li+)-containing treatments that can exert severe side effects. One target of Li+ is inositol monophosphatase-1 (IMPase1); inhibition of IMPase1 through small-molecule compounds may provide an alternative treatment for bipolar disorder. One such compound is the anti-inflammatory drug ebselen, which is well tolerated and safe; however, ebselen's exact mechanism of action in IMPase1 inhibition is not fully understood, preventing rational design of IMPase1 inhibitors. To fill this gap, we performed crystallographic and biochemical studies to investigate how ebselen inhibits IMPase1. We obtained a structure of IMPase1 in space group P21 after treatment with ebselen that revealed three key active-site loops (residues 33-44, 70-79, and 161-165) that are either disordered or in multiple conformations, supporting a hypothesis whereby dynamic conformational changes may be important for catalysis and ebselen inhibition. Using the thermal shift assay, we confirmed that ebselen significantly destabilizes the enzyme. Molecular docking suggests that ebselen could bind in the vicinity of His217. Investigation of the role of IMPase1 residues His217 and Cys218 suggests that inhibition of IMPase1 by ebselen may not be mediated via covalent modification of the active-site cysteine (Cys218) and is not affected by the covalent modification of other cysteine residues in the structure. Our results suggest that effects previously ascribed to ebselen-dependent inhibition likely result from disruption of essential active-site architecture, preventing activation of the IMPase1-Mg2+ complex.Communicated by Ramaswamy H. Sarma.
Assuntos
Cisteína , Compostos Organosselênicos , Humanos , Simulação de Acoplamento Molecular , Monoéster Fosfórico Hidrolases/química , Lítio/farmacologia , Lítio/uso terapêutico , Compostos Organosselênicos/farmacologia , Compostos Organosselênicos/químicaRESUMO
Cleome rutidosperma DC, commonly known in Jamaica as 'consumption-weed' is a plant traditionally used in folklore for treating tuberculosis and other infectious and chronic ailments. We evaluate for the first time the chemical composition and biological activities of the essential oil components of the complete aerial parts of this plant. The essential oil obtained by steam distillation (0.02%) was analyzed by a combination of gas chromatography-flame ionization detector (GC-FID), gas chromatography-mass spectroscopy (GC-MS) and retention index (RI). The volatile oil of C. rutidosperma was dominated by oxygenated diterpenes (67.6%); with (Z)-phytol (65.1%) being the single most abundant constituent. C. rutidosperma aerial essential oil exhibited moderate inhibition against the activity of recombinant arylamine N-acetyltransferase (NAT) from Mycobacterium marinum (IC50 22.20⯱â¯1.80⯵g/µL), while, racemic phytol had an inhibition with an IC50 of 22.33⯵g/µL⯱â¯0.50⯵g/µL, thus accounting for the NAT inhibition imparted by the crude oil. Inhibition of NAT, a key enzyme in mycobacterial growth may be the pathway in which phytol, shown in this study to interact with the active site using in-silico methods, renders its previously demonstrated anti-tubercular properties. The phytol rich essential oil also demonstrated antimicrobial activity against all nine human pathogenic bacteria and the fungus strain assayed, with the most significant inhibitory activity against Bacillus cereus and justifies the need for further evaluation and development of the essential oils from this plant.
Assuntos
Cleome/química , Óleos Voláteis/química , Óleos de Plantas/química , Bactérias/efeitos dos fármacos , Fungos/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Jamaica , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Óleos Voláteis/farmacologia , Óleos de Plantas/farmacologiaRESUMO
The effect of apocynin on the activity of arylamine N-acetyltransferases (NATs) in excised liver samples was examined using eighteen Sprague-Dawley rats. Three groups of six animals each were fed a normal diet alone or a treatment of 50 or 100 mg/kg/day of apocynin via gavages for eight (8) weeks. Chronic in vivo administration of apocynin led to significant (p < 0.001) reduction of in vitro liver NAT activity up to 93% as compared with untreated rats (18.80 ± 2.10 µmols p-anisidine/min/µg liver protein). In vitro exposure of untreated liver homogenates to apocynin led to a dose-dependent inhibition of NAT activity with IC50 = 0.69 ± 0.02 mM. In silico modelling of apocynin tautomers and radical species into human NAT crystal structures supported the hypothesis that thiol functionalities in NAT enzymes may be crucial in apocynin binding. The involvement of human NAT enzymes in different pathological conditions, such as cancer, has encouraged the research for selective NAT inhibitors in both humans and animal models with possible chemopreventive properties.
Assuntos
Acetofenonas/química , Antineoplásicos/química , Arilamina N-Acetiltransferase/antagonistas & inibidores , Inibidores Enzimáticos/química , Acetofenonas/metabolismo , Administração Oral , Sequência de Aminoácidos , Animais , Antineoplásicos/metabolismo , Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Domínio Catalítico , Misturas Complexas/química , Inibidores Enzimáticos/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Fígado/química , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Camundongos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
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.
Assuntos
Arilamina N-Acetiltransferase/metabolismo , Isoenzimas/metabolismo , Aminas/farmacologia , Animais , Arilamina N-Acetiltransferase/antagonistas & inibidores , Arilamina N-Acetiltransferase/genética , Humanos , Hidrazinas/farmacologia , Isoenzimas/antagonistas & inibidores , Camundongos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Especificidade por SubstratoRESUMO
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.
Assuntos
Acetil-CoA Hidrolase/metabolismo , Acetiltransferases/metabolismo , Arilamina N-Acetiltransferase/metabolismo , Ácido Fólico/metabolismo , Isoenzimas/metabolismo , Acetilação , Animais , Humanos , Hidrólise , Camundongos , Ressonância Magnética Nuclear BiomolecularRESUMO
A naphthoquinone inhibitor of human arylamine N-acetyltransferase 1 (hNAT1), a potential cancer biomarker and therapeutic target, has been reported which undergoes a distinctive concomitant color change from red to blue upon binding to the enzyme. Here we describe the use of in silico modeling alongside structure-activity relationship studies to advance the hit compound towards a potential probe to quantify hNAT1 levels in tissues. Derivatives with both a fifty-fold higher potency against hNAT1 and a two-fold greater absorption coefficient compared to the initial hit have been synthesized; these compounds retain specificity for hNAT1 and its murine homologue mNat2 over the isoenzyme hNAT2. A relationship between pKa, inhibitor potency and colorimetric properties has also been uncovered. The high potency of representative examples against hNAT1 in ZR-75-1 cell extracts also paves the way for the development of inhibitors with improved intrinsic sensitivity which could enable detection of hNAT1 in tissue samples and potentially act as tools for elucidating the unknown role hNAT1 plays in ER+ breast cancer; this could in turn lead to a therapeutic use for such inhibitors.
Assuntos
Arilamina N-Acetiltransferase/antagonistas & inibidores , Biomarcadores Tumorais/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , Naftoquinonas/farmacologia , Arilamina N-Acetiltransferase/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Colorimetria , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Isoenzimas/metabolismo , Modelos Moleculares , Estrutura Molecular , Naftoquinonas/síntese química , Naftoquinonas/química , Relação Estrutura-AtividadeRESUMO
Human arylamine N-acetyltransferase 1 (hNAT1) has become an attractive potential biomarker for estrogen-receptor-positive breast cancers. We describe here the mechanism of action of a selective non-covalent colorimetric biosensor for the recognition of hNAT1 and its murine homologue, mNat2, over their respective isoenzymes, leading to new opportunities in diagnosis. On interaction with the enzyme, the naphthoquinone probe undergoes an instantaneous and striking visible color change from red to blue. Spectroscopic, chemical, molecular modelling and biochemical studies reported here show that the color change is mediated by selective recognition between the conjugate base of the sulfonamide group within the probe and the conjugate acid of the arginine residue within the active site of both hNAT1 and mNat2. This represents a new mechanism for selective biomarker sensing and may be exploited as a general approach to the specific detection of biomarkers in disease.
Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Biomarcadores Tumorais/química , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Cor , Isoenzimas/química , Isoenzimas/metabolismo , Naftoquinonas/química , Naftoquinonas/metabolismo , Animais , Domínio Catalítico , Feminino , Humanos , Camundongos , Ligação ProteicaRESUMO
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.
Assuntos
Arilamina N-Acetiltransferase/genética , Preparações Farmacêuticas/metabolismo , Animais , Arilamina N-Acetiltransferase/antagonistas & inibidores , Arilamina N-Acetiltransferase/metabolismo , Inibidores Enzimáticos , Regulação Enzimológica da Expressão Gênica , HumanosRESUMO
Arylamine N-acetyltransferases (NATs) are phase II xenobiotic metabolizing enzymes playing a key role in the detoxification and metabolic activation of aromatic amine xenobiotics. The triennial International NAT Workshop has been an important academic meeting where developments in the study of NATs and aromatic amine metabolism have been presented. The 2010 Workshop took place in University Paris Diderot Paris, France. Topics included: structures and functions of eukaryotic and prokaryotic NATs, gene regulation and expression of human NATs, polymorphisms and their effects, arylamine metabolism and toxicity. Nomenclature issues were also discussed.
Assuntos
Arilamina N-Acetiltransferase/genética , Aminoácidos Aromáticos/metabolismo , Animais , Arilamina N-Acetiltransferase/metabolismo , Arilamina N-Acetiltransferase/fisiologia , Bactérias/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Polimorfismo Genético/genética , Xenobióticos/metabolismoRESUMO
Azoreductases are well known for azo pro-drug activation by gut flora. We show that azoreductases have a wider role in drug metabolism than previously thought as they can also reduce and hence activate nitrofurazone. Nitrofurazone, a nitroaromatic drug, is a broad spectrum antibiotic which has until now been considered as activated in bacteria by nitroreductases. The structure of the azoreductase with nitrofurazone bound was solved at 2.08 Å and shows nitrofurazone in an active conformation. Based on the structural information, the kinetics and stoichiometry of nitrofurazone reduction by azoreductase from P. aeruginosa, we propose a mechanism of activation which accounts for the ability of azoreductases to reduce both azo and nitroaromatic drugs. This mode of activation can explain the cytotoxic side-effects of nitrofurazone through human azoreductase homologues.
Assuntos
NADH NADPH Oxirredutases/metabolismo , Nitrofurazona/farmacocinética , Biotransformação , Modelos Moleculares , NADH NADPH Oxirredutases/química , Nitrofurazona/química , NitrorredutasesRESUMO
Azoreductases are important due to their ability to activate anti-inflammatory azo pro-drugs and to detoxify azo dyes. Three genes encoding azoreductases have been identified in Pseudomonas aeruginosa. We describe here a comparison of the three enzymes. The pure recombinant proteins each have a distinct substrate specificity profile against a range of azo substrates. Using the structure of P. aeruginosa azoreductase (paAzoR) 1 and the homology models of paAzoR2 and paAzoR3, we have identified residues important for substrate specificity. We have defined a novel flavin mononucleotide binding cradle, which is a recurrent motif in many flavodoxin-like proteins. A novel structure of paAzoR1 with the azo pro-drug balsalazide bound within the active site was determined by X-ray crystallography and demonstrates that the substrate is present in a hydrazone tautomer conformation. We propose that the structure with balsalazide bound represents an enzyme intermediate and, together with the flavin mononucleotide binding cradle, we propose a novel catalytic mechanism.
Assuntos
Compostos Azo/química , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , NADH NADPH Oxirredutases/genética , Nitrorredutases , Oxirredução , Pseudomonas aeruginosa/enzimologia , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
The identification, synthesis, and evaluation of a series of naphthoquinone derivatives as selective inhibitors of human arylamine N-acetyltransferase 1 and mouse arylamine N-acetyltransferase 2 are described. The compounds undergo a distinctive color change (red --> blue) upon binding to these human and mouse NAT isoenzymes driven by a proton transfer event. No color change is observed in the presence of functionally distinct but highly similar isoenzymes which are >70% identical. These molecules may be used as sensors to detect the presence of human NAT1 in cell lysates.
Assuntos
Arilamina N-Acetiltransferase/análise , Biomarcadores Tumorais/análise , Neoplasias da Mama/enzimologia , Colorimetria/métodos , Isoenzimas/análise , Animais , Arilamina N-Acetiltransferase/antagonistas & inibidores , Feminino , Humanos , Isoenzimas/antagonistas & inibidores , Camundongos , Modelos MolecularesRESUMO
Azoreductase 1 from Pseudomonas aeruginosa strain PAO1 (paAzoR1) catalyses the activation of the prodrug balsalazide and reduces the azo dye methyl red using reduced nicotinamide adenine dinucleotide cofactor as an electron donor. To investigate the mechanism of the enzyme, a Y131F mutation was introduced and the enzymic properties of the mutant were compared with those of the wild-type enzyme. The crystallographic structure of the mutant with methyl red bound was solved at 2.1 A resolution and compared with the wild-type structure. Tyr131 is important in the architecture of the active site but is not essential for enzymic activity.
Assuntos
NADH NADPH Oxirredutases/química , Tirosina/química , Compostos Azo , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X , Estabilidade Enzimática , Temperatura Alta , Cinética , Mesalamina/metabolismo , Mutagênese Sítio-Dirigida , NAD/metabolismo , NADH NADPH Oxirredutases/metabolismo , Nitrorredutases , Fenil-Hidrazinas/metabolismo , Dobramento de Proteína , Pseudomonas aeruginosa/enzimologiaRESUMO
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 treatment of inflammatory bowel disease, releasing the active component 5-aminosalycilic acid. The bacterium P. aeruginosa has three azoreductase genes, paAzoR1, paAzoR2 and paAzoR3, which as recombinant enzymes have been shown to have different substrate specificities. The mechanism 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.
Assuntos
Benzoquinonas/metabolismo , NADH NADPH Oxirredutases/química , Pseudomonas aeruginosa/enzimologia , Domínio Catalítico , Estabilidade Enzimática , Evolução Molecular , Flavinas/química , Temperatura Alta , Cinética , Mesalamina/química , NAD/metabolismo , NADP/metabolismo , Nitrorredutases , Concentração Osmolar , Oxirredução , Fenil-Hidrazinas/química , Filogenia , Ligação Proteica , Espectrofotometria UltravioletaRESUMO
There is currently a shortage of chemical molecules that can be used as bioactive probes to study molecular targets and potentially as starting points for drug discovery. One inexpensive way to address this problem is to use computational methods to screen a comprehensive database of small molecules to discover novel structures that could lead to alternative and better bioactive probes. Despite that pleasing logic the results have been somewhat mixed. Here we describe a virtual screening technique based on ligand-receptor shape complementarity, Ultrafast Shape Recognition (USR). USR is specifically applied to identify novel inhibitors of arylamine N-acetyltransferases by computationally screening almost 700 million molecular conformers in a time- and resource-efficient manner. A small number of the predicted active compounds were purchased and tested obtaining a confirmed hit rate of 40 per cent which is an outstanding result for a prospective virtual screening.
Assuntos
Algoritmos , Arilamina N-Acetiltransferase/antagonistas & inibidores , Biologia Computacional/métodos , Descoberta de Drogas/métodos , Inibidores Enzimáticos/isolamento & purificação , Conformação Proteica , Bases de Dados de ProteínasRESUMO
The identification, synthesis and evaluation of a series of rhodanine and thiazolidin-2,4-dione derivatives as selective inhibitors of human arylamine N-acetyltransferase 1 and mouse arylamine N-acetyltransferase 2 is described. The most potent inhibitors identified have submicromolar activity and inhibit both the recombinant proteins and human NAT1 in ZR-75 cell lysates in a competitive manner. (1)H NMR studies on purified mouse Nat2 demonstrate that the inhibitors bind within the putative active site of the enzyme.
Assuntos
Arilamina N-Acetiltransferase/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Isoenzimas/antagonistas & inibidores , Rodanina/síntese química , Tiazolidinedionas/síntese química , Animais , Sítios de Ligação , Biomarcadores Tumorais/antagonistas & inibidores , Neoplasias da Mama/patologia , Inibidores Enzimáticos , Feminino , Humanos , Camundongos , Rodanina/farmacologia , Tiazolidinedionas/farmacologiaRESUMO
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.