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Edith Sim

Publications and source records attributed to Edith Sim.

31 records · Page 2Linked to original sources
PubMed2003

Synthesis and in vitro evaluation of novel small molecule inhibitors of bacterial arylamine N-acetyltransferases (NATs).

The synthesis and inhibitory activity of a series of 5-substituted-(1,1-dioxo-2,3-dihydro-1H-1 lambda(6)-benzo[e][1,2]thiazin-4-ylidene)-thiazolidine-2,4-dione derivatives as competitive inhibitors of recombinant bacterial arylamine-N-acetyltransferases (NATs) are described. The most potent NAT inhibitors are those that contain planar hydrophobic substituents on the sultam nitrogen.

Alkylation↗
PubMed2003

An approach to identifying novel substrates of bacterial arylamine N-acetyltransferases.

Arylamine N-acetyltransferases (NATs) catalyse the acetylation of arylamine, arylhydrazine and arylhydroxylamine substrates by acetyl Coenzyme A. NAT has been discovered in a wide range of eukaryotic and prokaryotic species. Although prokaryotic NATs have been implicated in xenobiotic metabolism, to date no endogenous role has been identified for the arylamine N-acetyl transfer reaction in prokaryotes. Investigating the substrate specificity of these enzymes is one approach to determining a possible endogenous role for prokaryotic NATs. We describe an accurate and efficient assay for NAT activity that is suitable for high-throughput screening of potential NAT ligands. This assay has been utilised to identify novel substrates for pure NAT from Salmonella typhimurium and Mycobacterium smegmatis which show a relationship between the lipophilicity of the arylamine and its activity as a substrate. The lipophilic structure/activity relationship observed is proposed to depend on the topology of the active site using docking studies of the crystal structures of these NAT isoenzymes. The evidence suggests an endogenous role of NAT in the protection of bacteria from aromatic and lipophilic toxins.

Acetylation↗
PubMed2003

NMR investigation of the catalytic mechanism of arylamine N-acetyltransferase from Salmonella typhimurium.

Arylamine N-acetyltransferases (NAT) are a family of enzymes found in both eucaryotes and procaryotes, which catalyse the N-acetylation of a range of arylamine and hydrazine drugs and carcinogenic arylamines, using acetyl Coenzyme A as a cofactor. Here we describe a nuclear magnetic resonance (NMR) investigation of the interaction of substrates with Salmonella typhimurium NAT. For solution NMR investigations, pure recombinant NAT from S. typhimurium was used at up to 0.1 mM. We demonstrate that a hydrazine substrate, isoniazid (INH), binds to the protein in the absence of the cofactor, acetyl CoA, and thereby suggest that even though the catalysis may follow a ping-pong pathway, ligand-enzyme interactions can occur in the absence of acetyl CoA.

Arylamine N-Acetyltransferase↗
PubMed2003

Structural investigation of mutant Mycobacterium smegmatis arylamine N-acetyltransferase: a model for a naturally occurring functional polymorphism in Mycobacterium tuberculosis arylamine N-acetyltransferase.

Arylamine N-acetyltransferase (NAT) acetylates the front-line anti-tuberculosis drug isoniazid (INH) and has been identified in Mycobacterium tuberculosis. A naturally occurring single nucleotide polymorphism (SNP) was recently found in the NAT gene in clinical isolates of M. tuberculosis. The nucleotide change from G-->A (619) produces an amino acid change Gly(207) Arg, which appears to reduce the activity of the NAT from M. tuberculosis (TBNAT). It has not been possible to generate sufficient soluble recombinant TBNAT for 3D structural studies. Therefore, Mycobacterium smegmatis NAT (SMNAT), which has 60% identity to TBNAT and has Gly at 207, was used as a model to investigate the possible structural effects of the G-->A 619 SNP. The mutant form of SMnat (SM207Rnat) was constructed by in vitro site-directed mutagenesis and was heterologously expressed with an N-terminal His tag in Escherichia coli, for comparison with the SMNAT. Both recombinant SMNATs were purified using Ni affinity chromatography and treated with thrombin to cleave the tag. Both proteins were produced with average yields of over 10 mg/L and were active. Substrate specificity and thermal stability of SM207RNAT were assessed and compared with the wild type SMNAT using kinetic assays and circular dichroism spectroscopy. SM207RNAT was crystallised and a data set of 2.00 A resolution was obtained. The SM207RNAT had different substrate specificities to the wild type protein and the 3D structures revealed that the Gly(207) Arg mutation caused slight changes in the orientation of His(203) in SMNAT.

Amino Acid Sequence↗
PubMed2003

Skeletal muscles express the xenobiotic-metabolizing enzyme arylamine N-acetyltransferase.

The human arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are enzymes responsible for the acetylation of many arylamines and hydrazines, thereby playing an important role in both detoxification and activation of many drugs and carcinogens. Both enzymes show polymorphisms but exhibit key differences in substrate selectivity and tissue expression. In the present study, reverse transcriptase-PCR, Western blotting, and immunohistochemistry were used to investigate the expression of the NATs in human skeletal muscle. Despite the presence of its mRNA, NAT2 enzyme level was below the limit of detection. In contrast, both NAT1 mRNA and enzyme were readily detected in fetal, newborn, and adult muscles. In addition, punctate cytoplasmic and perinuclear NAT1 immunostaining was observed in all tissue sections, the staining being more intense in the fetal tissue. High expression of NAT1 enzyme in fetal muscle was also suggested by Western blotting. Because skeletal muscle accounts for a large proportion of body mass, muscle NAT1 expression may contribute significantly to the total activity in the body. These results further support the involvement of skeletal muscle in the metabolism of xenobiotics.

Adult↗
PubMed2003

Arylamine N-acetyltransferase-1 is highly expressed in breast cancers and conveys enhanced growth and resistance to etoposide in vitro.

Comparative two-dimensional proteome analysis was used to identify proteins differentially expressed in multiple clinical normal and breast cancer tissues. One protein, the expression of which was elevated in invasive ductal and lobular breast carcinomas when compared with normal breast tissue, was arylamine N-acetyltransferase-1 (NAT-1), a Phase II drug-metabolizing enzyme. NAT-1 overexpression in clinical breast cancers was confirmed at the mRNA level and immunohistochemical analysis of NAT-1 in 108 breast cancer donors demonstrated a strong association of NAT-1 staining with estrogen receptor-positive tumors. Analysis of the effect of active NAT-1 overexpression in a normal luminal epithelial-derived cell line demonstrated enhanced growth properties and etoposide resistance relative to control cells. Thus, NAT-1 may not only play a role in the development of cancers through enhanced mutagenesis but may also contribute to the resistance of some cancers to cytotoxic drugs.

Arylamine N-Acetyltransferase↗
PubMed2002

The structure of arylamine N-acetyltransferase from Mycobacterium smegmatis--an enzyme which inactivates the anti-tubercular drug, isoniazid.

Arylamine N-acetyltransferases which acetylate and inactivate isoniazid, an anti-tubercular drug, are found in mycobacteria including Mycobacterium smegmatis and Mycobacterium tuberculosis. We have solved the structure of arylamine N-acetyltransferase from M. smegmatis at a resolution of 1.7 A as a model for the highly homologous NAT from M. tuberculosis. The fold closely resembles that of NAT from Salmonella typhimurium, with a common catalytic triad and domain structure that is similar to certain cysteine proteases. The detailed geometry of the catalytic triad is typical of enzymes which use primary alcohols or thiols as activated nucleophiles. Thermal mobility and structural variations identify parts of NAT which might undergo conformational changes during catalysis. Sequence conservation among eubacterial NATs is restricted to structural residues of the protein core, as well as the active site and a hinge that connects the first two domains of the NAT structure. The structure of M. smegmatis NAT provides a template for modelling the structure of the M. tuberculosis enzyme and for structure-based ligand design as an approach to designing anti-TB drugs.

Antitubercular Agents↗
PubMed2002

The COOH terminus of arylamine N-acetyltransferase from Salmonella typhimurium controls enzymic activity.

Arylamine N-acetyltransferases (NATs) are a homologous family of enzymes, which acetylate arylamines, arylhydroxylamines, and arylhydrazines by acetyl transfer from acetyl-coenzyme A (Ac-CoA) and are found in many organisms. NAT was first identified as the enzyme responsible for the inactivation of the anti-tubercular drug isoniazid in humans. The three-dimensional structure of NAT from Salmonella typhimurium has been resolved and shown to have three distinct domains and an active site catalytic triad composed of "Cys(69)-His(107)-Asp(122)," which is typical of hydrolytic enzymes such as the cysteine proteases. The crystal unit cell consists of a dimer of tetramers, with the C terminus of individual monomers juxtaposed. To investigate the function of the first two domains of full-length NAT from S. typhimurium and to investigate the role of the C terminus of NAT, truncation mutants were made with either the C-terminal undecapeptide or the entire third domain (85 amino acids) missing. Unlike the full-length NAT protein (281 amino acids), the truncation mutants of NAT from S. typhimurium are toxic when overexpressed intracellularly in Escherichia coli. Full-length NAT hydrolyses Ac-CoA but only in the presence of an arylamine substrate. Both truncation mutants, however, hydrolyze Ac-CoA even in the absence of arylamine substrate, illustrating that the C-terminal undecapeptide controls hydrolysis of Ac-CoA by NAT from S. typhimurium.

Amines↗
PubMed2002

Expression and purification of the rifamycin amide synthase, RifF, an enzyme homologous to the prokaryotic arylamine N-acetyltransferases.

The assembly of the polyketide backbone of rifamycin B by the type I rifamycin polyketide synthase, encoded by the rifA-rifE genes, is terminated by the product of the rifF gene, an amide synthase that releases the completed undecaketide as its macrocyclic lactam. The sequence of the RifF protein from Amycolatopsis mediterranei shows 26% identity and 40% homology with the members of the arylamine N-acetyltransferase (NAT) family of proteins. Based on the homology of the primary structures and the similarity of the reactions catalyzed by the two enzymes, we have compared the RifF protein with members of the NAT family. We have modeled the three-dimensional (3D) structure of RifF using NAT from Salmonella typhimurium and Mycobacterium smegmatis as a template. Proteolytic digestions of RifF revealed accessible regions in the protein which are in agreement with the modeled structure. We have expressed the whole protein and individual domains of the protein based on comparison with NAT from S. typhimurium and have purified the proteins by affinity chromatography using a hexahistidine tag. RifF has been further purified using ion-exchange (Mono Q) chromatography. An antiserum has been generated using the C-terminal nona- and tridecapeptides of RifF and has been shown to recognize RifF uniquely. It does not cross-react with any other member of the NAT family.

Amino Acid Sequence↗
PubMed2002

Identification and functional characterization of novel polymorphisms associated with the genes for arylamine N-acetyltransferases in mice.

Arylamine N-acetyltransferase (NAT) polymorphism in humans has been associated with variation in susceptibility to drug toxicity and cancer. In mice, three NAT isoenzymes are encoded by Nat1, Nat2 and Nat3 genes. Only Nat2 has been shown previously to be polymorphic, a single nucleotide substitution causing the slow acetylator phenotype in the A/J strain. We sequenced the Nat genes from inbred (CBA and 129/Ola), outbred (PO and TO) and wild-derived inbred (Mus spretus and Mus musculus castaneus) mouse strains and report polymorphism in all three Nat genes of M. spretus and in Nat2 and Nat3 genes of M. m. castaneus. Enzymatic activity assays using liver homogenates demonstrated that M. m. castaneus is a 'fast' and M. spretus a 'slow' acetylator. Western blot analysis indicated that hepatic NAT2 protein is less abundant in M. spretus than M. m. castaneus. The new allozymes were expressed in a mammalian cell line and NAT enzymatic activity was measured with a series of substrates. NAT1 and NAT2 isoenzymes of M. m. castaneus exhibited a higher rate of acetylation, compared with those of M. spretus. Activity of the NAT3 allozymes was hardly detectable, although the Nat3 gene does appear to be transcribed, since mRNA was detected by RT-PCR in the spleen. Additional polymorphisms, useful for Nat-related genetic studies, have been identified between BALB/c, C57Bl/6J, A/J, 129/Ola, CBA, PO, TO, M. m. castaneus and M. spretus strains in four microsatellite repeats located close to the Nat genes.

Animals↗
PubMed2002

The pharmacogenetics of NAT: structural aspects.

Arylamine N-acetyltransferases (NATs) catalyze the transfer of an acetyl group from acetyl-CoA to arylhydrazines and to arylamine drugs and carcinogens or to their N-hydroxylated metabolites. NAT plays an important role in detoxification and metabolic activation of xenobiotics and was first identified as the enzyme responsible for inactivation of the antitubercular drug isoniazid, an arylhydrazine. The rate of inactivation was polymorphically distributed in the population: the first example of interindividual pharmacogenetic variation. Polymorphism in NAT activity is primarily due to single nucleotide polymorphisms (SNPs) in the coding region of NAT genes. NAT enzymes are widely distributed in eukaryotes and genome sequences have revealed many homologous members of this enzyme family in prokaryotes. The structures of S almonella typhimurium and Mycobacterium smegmatis NATs have been determined, revealing a unique fold in which a catalytic triad (Cys-His-Asp) forms the active site. Determination of prokaryotic and eukaryotic NAT structures could lead to a better understanding of their role in xenobiotics and endogenous metabolism.

Alleles↗
PubMed2001

Eubacterial arylamine N-acetyltransferases - identification and comparison of 18 members of the protein family with conserved active site cysteine, histidine and aspartate residues.

Arylamine N-acetyltransferases (NATs) are enzymes involved in the detoxification of a range of arylamine and hydrazine-based xenobiotics. NATs have been implicated in the endogenous metabolism of p-aminobenzoyl glutamate in eukaryotes, although very little is known about the distribution and function of NAT in the prokaryotic kingdom. Using DNA library screening techniques and the analysis of data from whole-genome sequencing projects, we have identified 18 nat-like sequences from the Proteobacteria and Firmicutes. Recently, the three-dimensional structure of NAT derived from the bacterium Salmonella typhimurium (PDB accession code 1E2T) was resolved and revealed an active site catalytic triad composed of Cys(69)-His(107)-Asp(122). These residues have been shown to be conserved in all prokaryotic and eukaryotic NAT homologues together with three highly conserved regions which are found proximal to the active site triad. The characterization of prokaryotic NATs and NAT-like enzymes is reported. It is also predicted that prokaryotic NATs, based on gene cluster composition and distribution amongst genomes, participate in the metabolism of xenobiotics derived from decomposition of organic materials.

Amino Acid Motifs↗