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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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The downfield region of the 1D 1H NMR spectra of (A) mouse and (B) hamster Nat2 collected using a jump-return pulse sequence. These peaks arise from the indole HN of two tryptophan residues (peaks 2 and 3, Trp67 and Trp132 respectively) and from imidazole HN of histidine (peaks 1 and 4, His).
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fig6: The downfield region of the 1D 1H NMR spectra of (A) mouse and (B) hamster Nat2 collected using a jump-return pulse sequence. These peaks arise from the indole HN of two tryptophan residues (peaks 2 and 3, Trp67 and Trp132 respectively) and from imidazole HN of histidine (peaks 1 and 4, His).

Mentions: The downfield region of the 1H 1D spectra, collected with a jump-return sequence, of mouse and hamster Nat2 also show a similar pattern of peaks (Fig. 6A and B). The four peaks are compared in Table 4, together with their assignments. Two of the peaks (2 and 3) have been assigned previously for hamster Nat2 and can be assigned in the mouse spectrum by homology; these correspond to strongly hydrogen bonded tryptophan indole groups: the tryptophan associated with the P-loop (Trp132, see Fig. 7) and Trp67, which is located adjacent to the active-site Cys68, and appears to have a structural role. The strong hydrogen bonds are likely to be responsible for the large downfield shifts of these tryptophan peaks compared to random coil values (∼9.5 ppm). The two remaining peaks (1 and 4) are assigned to the imidazole 1HN of histidine on the basis of 15N decoupling experiments; these peaks have not been assigned previously for hamster Nat2. The side chain 1HN groups of histidine are usually not observed by NMR, particularly at pH 7, due to their rapid exchange with solvent protons. The observation of these peaks indicates that the histidine residues are probably buried within the protein and that the side chain 1HN are involved in hydrogen bonds. Analysis of the homology model of mouse Nat2 suggests that at least one of these peaks might arise from the active site histidine, His107. The homology model of mouse Nat 2 indicates that the side chain of His107 is involved in two hydrogen bonds in the active site and is the least accessible to solvent. In addition, recent enzymological studies with hamster Nat2 have confirmed that the active-site cysteine and histidine residues exist as a thiolate-imidazolium ion pair [35], in which the histidine has two tightly bound and H-bonded protons.


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)

The downfield region of the 1D 1H NMR spectra of (A) mouse and (B) hamster Nat2 collected using a jump-return pulse sequence. These peaks arise from the indole HN of two tryptophan residues (peaks 2 and 3, Trp67 and Trp132 respectively) and from imidazole HN of histidine (peaks 1 and 4, His).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: The downfield region of the 1D 1H NMR spectra of (A) mouse and (B) hamster Nat2 collected using a jump-return pulse sequence. These peaks arise from the indole HN of two tryptophan residues (peaks 2 and 3, Trp67 and Trp132 respectively) and from imidazole HN of histidine (peaks 1 and 4, His).
Mentions: The downfield region of the 1H 1D spectra, collected with a jump-return sequence, of mouse and hamster Nat2 also show a similar pattern of peaks (Fig. 6A and B). The four peaks are compared in Table 4, together with their assignments. Two of the peaks (2 and 3) have been assigned previously for hamster Nat2 and can be assigned in the mouse spectrum by homology; these correspond to strongly hydrogen bonded tryptophan indole groups: the tryptophan associated with the P-loop (Trp132, see Fig. 7) and Trp67, which is located adjacent to the active-site Cys68, and appears to have a structural role. The strong hydrogen bonds are likely to be responsible for the large downfield shifts of these tryptophan peaks compared to random coil values (∼9.5 ppm). The two remaining peaks (1 and 4) are assigned to the imidazole 1HN of histidine on the basis of 15N decoupling experiments; these peaks have not been assigned previously for hamster Nat2. The side chain 1HN groups of histidine are usually not observed by NMR, particularly at pH 7, due to their rapid exchange with solvent protons. The observation of these peaks indicates that the histidine residues are probably buried within the protein and that the side chain 1HN are involved in hydrogen bonds. Analysis of the homology model of mouse Nat2 suggests that at least one of these peaks might arise from the active site histidine, His107. The homology model of mouse Nat 2 indicates that the side chain of His107 is involved in two hydrogen bonds in the active site and is the least accessible to solvent. In addition, recent enzymological studies with hamster Nat2 have confirmed that the active-site cysteine and histidine residues exist as a thiolate-imidazolium ion pair [35], in which the histidine has two tightly bound and H-bonded protons.

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