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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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750 MHz 2D 1H-15N HSQC spectrum of mouse Nat2.
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fig5: 750 MHz 2D 1H-15N HSQC spectrum of mouse Nat2.

Mentions: NMR studies are ideal for investigating protein interactions with substrates and inhibitors in solution. Therefore, we have generated mouse Nat2 uniformly labelled with 15N to allow 2D NMR investigations of the protein. The 2D 1H-15N HSQC spectrum of mouse Nat2 is shown in Fig. 5; this spectrum contains a peak for each backbone amide (1HN-15N) and additional peaks from the side chains of Asn, Gln and Trp. The HSQC spectrum is well resolved, as expected for a compact globular structure, and some cross peaks are relatively sharp for a 30 kDa protein, this may indicate some flexibility between the domains of mouse Nat2 or the presence of mobile loops. The HSQC spectrum of mouse Nat2 has been compared with the corresponding spectrum from hamster Nat2 for which a full assignment is available [34]. The pattern of peaks in the HSQC spectra is extremely similar for the two homologous proteins; this allows some peaks observed in the mouse Nat2 spectrum to be assigned on the basis of the hamster Nat2 spectrum.


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)

750 MHz 2D 1H-15N HSQC spectrum of mouse Nat2.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: 750 MHz 2D 1H-15N HSQC spectrum of mouse Nat2.
Mentions: NMR studies are ideal for investigating protein interactions with substrates and inhibitors in solution. Therefore, we have generated mouse Nat2 uniformly labelled with 15N to allow 2D NMR investigations of the protein. The 2D 1H-15N HSQC spectrum of mouse Nat2 is shown in Fig. 5; this spectrum contains a peak for each backbone amide (1HN-15N) and additional peaks from the side chains of Asn, Gln and Trp. The HSQC spectrum is well resolved, as expected for a compact globular structure, and some cross peaks are relatively sharp for a 30 kDa protein, this may indicate some flexibility between the domains of mouse Nat2 or the presence of mobile loops. The HSQC spectrum of mouse Nat2 has been compared with the corresponding spectrum from hamster Nat2 for which a full assignment is available [34]. The pattern of peaks in the HSQC spectra is extremely similar for the two homologous proteins; this allows some peaks observed in the mouse Nat2 spectrum to be assigned on the basis of the hamster Nat2 spectrum.

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