Comparative analysis of xenobiotic metabolising N-acetyltransferases from ten non-human primates as in vitro models of human homologues

Xenobiotic metabolising N-acetyltransferases (NATs) perform biotransformation of drugs and carcinogens. Human NAT1 is associated with endogenous metabolic pathways of cells and is a candidate drug target for cancer. Human NAT2 is a well-characterised polymorphic xenobiotic metabolising enzyme, modulating susceptibility to drug-induced toxicity. Human NATs are difficult to express to high purification yields, complicating large-scale production for high-throughput screens or use in sophisticated enzymology assays and crystallography. We undertake comparative functional investigation of the NAT homologues of ten non-human primates, to characterise their properties and evaluate their suitability as models of human NATs. Considering the amount of generated recombinant protein, the enzymatic activity and thermal stability, the NAT homologues of non-human primates are demonstrated to be a much more effective resource for in vitro studies compared with human NATs. Certain NAT homologues are proposed as better models, such as the NAT1 of macaques Macaca mulatta and M. sylvanus, the NAT2 of Erythrocebus patas, and both NAT proteins of the gibbon Nomascus gabriellae which show highest homology to human NATs. This comparative investigation will facilitate in vitro screens towards discovery and optimisation of candidate pharmaceutical compounds for human NAT isoenzymes, while enabling better understanding of NAT function and evolution in primates

Mapping the genes for arylamine N-acetyltransferases

Arylamine N-acetyltransferases are xenobiotic metabolising enzymes, involved in metabolic pathways leading to either detoxification or bio-activation of drugs and carcinogens. In humans, polymorphisms of the two functional NAT genes (NAT1 and NAT2} have been implicated to cancer susceptibility. The genomic region of the NAT1 loci, 8p22, is often deleted in tumours and is believed to contain tumour suppressor genes. Two previously developed cosmid probes, carrying either NAT1 or NAT2, were tested as probes for FISH analysis using nuclei from healthy lymphocytes and the RT112 urothelial carcinoma cell line (Chapter 3). FISH analysis of cells obtained from barbotage samples of bladder cancer patients was performed and deletion of the NAT genomic region was a common observation (Chapter 6). To allow refined mapping of the NAT region on 8p22, a series of PAC clones carrying the NAT loci were isolated by PCR screening of a genomic library (Chapter 3). Furthermore, the NAT1 and NAT2 isoenzymes were studied in healthy intestinal and mammary tissue, as a first step towards understanding their potential involvement in these two types of cancer (Chapter 6). The mouse has been extensively used as a model for studying NAT. Three functional Nat genes are present in the mouse genome. A fine restriction map was generated for the Nat1 and Nat2 genomic region in mice, using Nat-positive genomic DNA plasmid clones previously isolated from the fast acetylator Balb/c and 129/Ola strains. The Nat1 and Nat2 genes were mapped 9.4kb apart and no polymorphisms were detected between the two strains for the restriction enzymes used for mapping (Chapter 4). The 129/Ola restriction map has since been the basis for the production of a construct for Nat2 knockout and transgenic mouse strains. PAC clones positive for Nat were isolated from a mouse genomic library and used as FISH probes to map the three murine Nat genes on chromosome 8, band B3.1-3.3. One PAC clone contained all three Nat loci, establishing co-localisation of Nat3 with the other two Nat genes in mice. The minimum distance of Nat3 from Nat1 and Nat2 was estimated to be 22kb, while the three loci are within 130kb. YAC clones carrying the Nat genes were also isolated to facilitate physical mapping of the Nat cluster in mice (Chapter 5). This will allow integration of cytogenetic, physical and previous genetic data for comparative studies

Mapping the genes for arylamine N-acetyltransferases

Arylamine N-acetyltransferases are xenobiotic metabolising enzymes, involved in metabolic pathways leading to either detoxification or bio-activation of drugs and carcinogens. In humans, polymorphisms of the two functional NAT genes (NAT1 and NAT2} have been implicated to cancer susceptibility. The genomic region of the NAT1 loci, 8p22, is often deleted in tumours and is believed to contain tumour suppressor genes. Two previously developed cosmid probes, carrying either NAT1 or NAT2, were tested as probes for FISH analysis using nuclei from healthy lymphocytes and the RT112 urothelial carcinoma cell line (Chapter 3). FISH analysis of cells obtained from barbotage samples of bladder cancer patients was performed and deletion of the NAT genomic region was a common observation (Chapter 6). To allow refined mapping of the NAT region on 8p22, a series of PAC clones carrying the NAT loci were isolated by PCR screening of a genomic library (Chapter 3). Furthermore, the NAT1 and NAT2 isoenzymes were studied in healthy intestinal and mammary tissue, as a first step towards understanding their potential involvement in these two types of cancer (Chapter 6). The mouse has been extensively used as a model for studying NAT. Three functional Nat genes are present in the mouse genome. A fine restriction map was generated for the Nat1 and Nat2 genomic region in mice, using Nat-positive genomic DNA plasmid clones previously isolated from the fast acetylator Balb/c and 129/Ola strains. The Nat1 and Nat2 genes were mapped 9.4kb apart and no polymorphisms were detected between the two strains for the restriction enzymes used for mapping (Chapter 4). The 129/Ola restriction map has since been the basis for the production of a construct for Nat2 knockout and transgenic mouse strains. PAC clones positive for Nat were isolated from a mouse genomic library and used as FISH probes to map the three murine Nat genes on chromosome 8, band B3.1-3.3. One PAC clone contained all three Nat loci, establishing co-localisation of Nat3 with the other two Nat genes in mice. The minimum distance of Nat3 from Nat1 and Nat2 was estimated to be 22kb, while the three loci are within 130kb. YAC clones carrying the Nat genes were also isolated to facilitate physical mapping of the Nat cluster in mice (Chapter 5). This will allow integration of cytogenetic, physical and previous genetic data for comparative studies.</p

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

Current trends in N-acetyltransferase research arising from the 2007 International NAT Workshop

Arylamine N-acetyltransferase (NAT) research has been influenced in recent years by the rapid progress in genomics, proteomics, structural genomics and other cutting-edge disciplines. To keep up with these advancements, the NAT scientific community has fostered collaboration and exchange of know-how between its members. As a specialized event bringing together experts from many different laboratories, the triennial International NAT Workshop has been instrumental in maintaining this culture over the past ten years. The 2007 Workshop took place in Alexandroupolis, Greece, and covered ongoing research on the structure and enzymatic function of human NATs, the prokaryotic and eukaryotic models for NAT, the mechanisms of NAT gene regulation and expression, the frequencies and effects of polymorphisms in the human NAT genes, and the involvement of NATs in multifactorial diseases, including cancer, allergic conditions, endometriosis and endemic nephropathies. Gene nomenclature issues were also addressed and the participants discussed current trends in the field

Joint Analysis of Phenotypic and Genomic Diversity Sheds Light on the Evolution of Xenobiotic Metabolism in Humans

Variation in genes involved in the absorption, distribution, metabolism, and excretion of drugs (ADME) can influence individual response to a therapeutic treatment. The study of ADME genetic diversity in human populations has led to evolutionary hypotheses of adaptation to distinct chemical environments. Population differentiation in measured drug metabolism phenotypes is, however, scarcely documented, often indirectly estimated via genotype-predicted phenotypes. We administered seven probe compounds devised to target six cytochrome P450 enzymes and the P-glycoprotein (P-gp) activity to assess phenotypic variation in four populations along a latitudinal transect spanning over Africa, the Middle East, and Europe (349 healthy Ethiopian, Omani, Greek, and Czech volunteers). We demonstrate significant population differentiation for all phenotypes except the one measuring CYP2D6 activity. Genome-wide association studies (GWAS) evidenced that the variability of phenotypes measuring CYP2B6, CYP2C9, CYP2C19, and CYP2D6 activity was associated with genetic variants linked to the corresponding encoding genes, and additional genes for the latter three. Instead, GWAS did not indicate any association between genetic diversity and the phenotypes measuring CYP1A2, CYP3A4, and P-gp activity. Genome scans of selection highlighted multiple candidate regions, a few of which included ADME genes, but none overlapped with the GWAS candidates. Our results suggest that different mechanisms have been shaping the evolution of these phenotypes, including phenotypic plasticity, and possibly some form of balancing selection. We discuss how these contrasting results highlight the diverse evolutionary trajectories of ADME genes and proteins, consistent with the wide spectrum of both endogenous and exogenous molecules that are their substrates.</p

Fusarium verticillioides NAT1 ( FDB2 ) N ‐malonyltransferase is structurally, functionally and phylogenetically distinct from its N ‐acetyltransferase ( NAT ) homologues

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl-CoA-dependent enzyme homologous to xenobiotic metabolizing arylamine N-acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N-malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N-acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two “tunnel-like” entries separated by a “bridge-like” helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N-terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl-CoA better than malonyl-CoA, dimerization changes the active site to allow malonyl-CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl-group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl-group, acetyl-CoA cannot form such stabilizing interactions, while longer acyl-CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control