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Computational and experimental analyses of mammalian arylamine N-acetyltransferase structure and function.

ABSTRACT: Arylamine N-acetyltransferases (NATs) play an important role in the metabolism of arylamine and hydrazine drugs and many arylamine procarcinogens. The two human N-acetyltransferases, NAT1 and NAT2, are widely distributed in human tissues and are highly polymorphic. Although many xenobiotic procarcinogens and drugs are known mammalian NAT substrates, it is unclear what physiological roles these enzymes might play, what endogenous substrates they primarily act upon, or the mechanisms underlying the functional effects of specific NAT gene coding region single-nucleotide polymorphisms. Analyses of mammalian NAT protein structures can greatly help to answer these questions. Homology modeling techniques can be used to approximate mammalian NAT structures using known bacterial NAT crystal structures as templates. In comparison to the bacterial template NATs used for homology modeling, mammalian NATs have a 17-residue insert of unknown structure and function. Homology modeling analyses yielded two different alignments (Modeler 8v1 or 3DCoffee algorithms) that placed this insert in two likely alternative locations. Secondary structure prediction techniques and experimental analyses of a series of human NAT2 mutants with artificial deletions/replacements of the insert region distinguished one of these alternatives as the most likely insert location and provided a better understanding of its structure and function. This study demonstrates both the utility and limitations of computational structural modeling with proteins that differ as much as the mammalian and bacterial NATs.

SUBMITTER: Walraven JM

PROVIDER: S-EPMC2085365 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Computational and experimental analyses of mammalian arylamine N-acetyltransferase structure and function.

Walraven Jason M JM Trent John O JO Hein David W DW

Drug metabolism and disposition: the biological fate of chemicals 20070319 6

Arylamine N-acetyltransferases (NATs) play an important role in the metabolism of arylamine and hydrazine drugs and many arylamine procarcinogens. The two human N-acetyltransferases, NAT1 and NAT2, are widely distributed in human tissues and are highly polymorphic. Although many xenobiotic procarcinogens and drugs are known mammalian NAT substrates, it is unclear what physiological roles these enzymes might play, what endogenous substrates they primarily act upon, or the mechanisms underlying th ...[more]

PMID: 17371801

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Project description: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.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.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.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.

Dataset Information

Computational and experimental analyses of mammalian arylamine N-acetyltransferase structure and function.

Publications

Computational and experimental analyses of mammalian arylamine N-acetyltransferase structure and function.

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