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Mouse N-acetyltransferase type 2, the homologue of human N-acetyltransferase type 1.

Kawamura A, Westwood I, Wakefield L, Long H, Zhang N, Walters K, Redfield C, Sim E - Biochem. Pharmacol. (2008)

Bottom Line: In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids.We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2.We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.

ABSTRACT
There is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

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Related in: MedlinePlus

Titration of mouse Nat2 with bisphenol A. The magnitudes of changes in chemical shift observed for the four downfield shifted peaks are shown as a function of the amount of added bisphenol A. The largest shifts are observed for peaks 1 (filled circle) and 4 (open square) which arise from histidine; smaller shifts are observed for peaks 2 (open triangle) and 3 (filled triangle).
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fig9: Titration of mouse Nat2 with bisphenol A. The magnitudes of changes in chemical shift observed for the four downfield shifted peaks are shown as a function of the amount of added bisphenol A. The largest shifts are observed for peaks 1 (filled circle) and 4 (open square) which arise from histidine; smaller shifts are observed for peaks 2 (open triangle) and 3 (filled triangle).

Mentions: In order to probe the molecular interactions of mouse Nat2 and the exogenous steroid inhibitors, we have investigated the effect of bisphenol A on the 1D 1H NMR spectrum of mouse Nat2 (Figs. 8 and 9). The observation of changes in chemical shift for particular amino acid residues which result from the addition of a ligand are usually interpreted as an indication that these residues are located in close proximity to the ligand-binding site. When bisphenol A was titrated into a solution of mouse Nat2, the two downfield histidine peaks (1 and 4, Table 4) were observed to shift by more than 0.05 ppm (Fig. 9); peak 1 shifts downfield by ∼0.12 ppm and peak 4 shifts upfield by ∼0.08 ppm in the presence of 8 equivalents of bisphenol A. Additional small shifts were also observed for the side chain HN of Trp67 and Trp132 (Fig. 9). The observation of progressive changes in chemical shift upon addition of bisphenol A indicates fast exchange on the NMR timescale; this would be expected given the relatively weak affinity of mouse Nat2 for this ligand (IC50 = 290 ± 10 μM). We have postulated above that at least one of the histidine peaks arises from the active-site histidine, His107; therefore, these results show that binding of bisphenol A is likely to take place in close proximity to the active site of mouse Nat2.


Mouse N-acetyltransferase type 2, the homologue of human N-acetyltransferase type 1.

Kawamura A, Westwood I, Wakefield L, Long H, Zhang N, Walters K, Redfield C, Sim E - Biochem. Pharmacol. (2008)

Titration of mouse Nat2 with bisphenol A. The magnitudes of changes in chemical shift observed for the four downfield shifted peaks are shown as a function of the amount of added bisphenol A. The largest shifts are observed for peaks 1 (filled circle) and 4 (open square) which arise from histidine; smaller shifts are observed for peaks 2 (open triangle) and 3 (filled triangle).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2279149&req=5

fig9: Titration of mouse Nat2 with bisphenol A. The magnitudes of changes in chemical shift observed for the four downfield shifted peaks are shown as a function of the amount of added bisphenol A. The largest shifts are observed for peaks 1 (filled circle) and 4 (open square) which arise from histidine; smaller shifts are observed for peaks 2 (open triangle) and 3 (filled triangle).
Mentions: In order to probe the molecular interactions of mouse Nat2 and the exogenous steroid inhibitors, we have investigated the effect of bisphenol A on the 1D 1H NMR spectrum of mouse Nat2 (Figs. 8 and 9). The observation of changes in chemical shift for particular amino acid residues which result from the addition of a ligand are usually interpreted as an indication that these residues are located in close proximity to the ligand-binding site. When bisphenol A was titrated into a solution of mouse Nat2, the two downfield histidine peaks (1 and 4, Table 4) were observed to shift by more than 0.05 ppm (Fig. 9); peak 1 shifts downfield by ∼0.12 ppm and peak 4 shifts upfield by ∼0.08 ppm in the presence of 8 equivalents of bisphenol A. Additional small shifts were also observed for the side chain HN of Trp67 and Trp132 (Fig. 9). The observation of progressive changes in chemical shift upon addition of bisphenol A indicates fast exchange on the NMR timescale; this would be expected given the relatively weak affinity of mouse Nat2 for this ligand (IC50 = 290 ± 10 μM). We have postulated above that at least one of the histidine peaks arises from the active-site histidine, His107; therefore, these results show that binding of bisphenol A is likely to take place in close proximity to the active site of mouse Nat2.

Bottom Line: In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids.We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2.We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom.

ABSTRACT
There is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.

Show MeSH
Related in: MedlinePlus