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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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(a) and (b) The structure of mouse Nat2. The mouse Nat2 protein structure was produced by homology modelling with the program Modeller 8v2 [33]. The human NAT1 crystal structure (PDB code 2IJA) was used as a template, and the amino acid sequences of human NAT1 and mouse Nat2 were aligned with the program ClustalW [48]. The mouse Nat2 ribbon structure is shown in green. The ribbon structures of the inter-domain loop region and C-terminal hexapeptide residues (residues 168–184 and 285–290) are shown in dark green, and the active site catalytic triad (Cys68, His107 and Asp122) residues are shown in ball and stick representation. The pdb file of the mouse Nat2 homology model is available upon request. The figure was produced with Aesop, as previously described [49]. (c) and (d) A comparison of the human NAT1 crystal structure (PDB code 2IJA) and the mouse Nat2 homology model. The human NAT1 crystal structure is shown in blue and the mouse Nat2 model is shown in green. The two proteins share 82% identity at the amino acid level. Over 1191 equivalent atoms, the two structures have a root mean squared deviation of 0.75 Å. The catalytic triad residues for both proteins are shown in ball and stick representation. The views in (b) and (d) were obtained by rotating the structures shown in (a) and (c) by 30°. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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fig4: (a) and (b) The structure of mouse Nat2. The mouse Nat2 protein structure was produced by homology modelling with the program Modeller 8v2 [33]. The human NAT1 crystal structure (PDB code 2IJA) was used as a template, and the amino acid sequences of human NAT1 and mouse Nat2 were aligned with the program ClustalW [48]. The mouse Nat2 ribbon structure is shown in green. The ribbon structures of the inter-domain loop region and C-terminal hexapeptide residues (residues 168–184 and 285–290) are shown in dark green, and the active site catalytic triad (Cys68, His107 and Asp122) residues are shown in ball and stick representation. The pdb file of the mouse Nat2 homology model is available upon request. The figure was produced with Aesop, as previously described [49]. (c) and (d) A comparison of the human NAT1 crystal structure (PDB code 2IJA) and the mouse Nat2 homology model. The human NAT1 crystal structure is shown in blue and the mouse Nat2 model is shown in green. The two proteins share 82% identity at the amino acid level. Over 1191 equivalent atoms, the two structures have a root mean squared deviation of 0.75 Å. The catalytic triad residues for both proteins are shown in ball and stick representation. The views in (b) and (d) were obtained by rotating the structures shown in (a) and (c) by 30°. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Mentions: We have generated a structural model of the mouse Nat2 protein, whose amino acid sequence is over 80% identical to human NAT1, by using the program Modeller 8v2 [33]. Fig. 4 shows the structural model of mouse Nat2 and a comparison of the mouse Nat2 and human NAT1 protein structures. The two structures share 1191 equivalent atoms, over which the root mean squared deviation is 0.75 Å. In mouse Nat2, as in human NAT1, the inter-domain loop and the C-terminus occlude the active site. Despite the evidence for coenzyme A binding to human NAT2 without major conformational rearrangement of the C-terminus and inter-domain loop regions, it has not been possible to perform ligand docking into the mouse Nat2 or human NAT1 structures without disturbing these regions of the protein structure. Therefore, it remains possible that the inter-domain loop regions and C-termini of the eukaryotic NAT proteins are conformationally flexible, in order to accommodate substrate or inhibitor binding.


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)

(a) and (b) The structure of mouse Nat2. The mouse Nat2 protein structure was produced by homology modelling with the program Modeller 8v2 [33]. The human NAT1 crystal structure (PDB code 2IJA) was used as a template, and the amino acid sequences of human NAT1 and mouse Nat2 were aligned with the program ClustalW [48]. The mouse Nat2 ribbon structure is shown in green. The ribbon structures of the inter-domain loop region and C-terminal hexapeptide residues (residues 168–184 and 285–290) are shown in dark green, and the active site catalytic triad (Cys68, His107 and Asp122) residues are shown in ball and stick representation. The pdb file of the mouse Nat2 homology model is available upon request. The figure was produced with Aesop, as previously described [49]. (c) and (d) A comparison of the human NAT1 crystal structure (PDB code 2IJA) and the mouse Nat2 homology model. The human NAT1 crystal structure is shown in blue and the mouse Nat2 model is shown in green. The two proteins share 82% identity at the amino acid level. Over 1191 equivalent atoms, the two structures have a root mean squared deviation of 0.75 Å. The catalytic triad residues for both proteins are shown in ball and stick representation. The views in (b) and (d) were obtained by rotating the structures shown in (a) and (c) by 30°. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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Related In: Results  -  Collection

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

fig4: (a) and (b) The structure of mouse Nat2. The mouse Nat2 protein structure was produced by homology modelling with the program Modeller 8v2 [33]. The human NAT1 crystal structure (PDB code 2IJA) was used as a template, and the amino acid sequences of human NAT1 and mouse Nat2 were aligned with the program ClustalW [48]. The mouse Nat2 ribbon structure is shown in green. The ribbon structures of the inter-domain loop region and C-terminal hexapeptide residues (residues 168–184 and 285–290) are shown in dark green, and the active site catalytic triad (Cys68, His107 and Asp122) residues are shown in ball and stick representation. The pdb file of the mouse Nat2 homology model is available upon request. The figure was produced with Aesop, as previously described [49]. (c) and (d) A comparison of the human NAT1 crystal structure (PDB code 2IJA) and the mouse Nat2 homology model. The human NAT1 crystal structure is shown in blue and the mouse Nat2 model is shown in green. The two proteins share 82% identity at the amino acid level. Over 1191 equivalent atoms, the two structures have a root mean squared deviation of 0.75 Å. The catalytic triad residues for both proteins are shown in ball and stick representation. The views in (b) and (d) were obtained by rotating the structures shown in (a) and (c) by 30°. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
Mentions: We have generated a structural model of the mouse Nat2 protein, whose amino acid sequence is over 80% identical to human NAT1, by using the program Modeller 8v2 [33]. Fig. 4 shows the structural model of mouse Nat2 and a comparison of the mouse Nat2 and human NAT1 protein structures. The two structures share 1191 equivalent atoms, over which the root mean squared deviation is 0.75 Å. In mouse Nat2, as in human NAT1, the inter-domain loop and the C-terminus occlude the active site. Despite the evidence for coenzyme A binding to human NAT2 without major conformational rearrangement of the C-terminus and inter-domain loop regions, it has not been possible to perform ligand docking into the mouse Nat2 or human NAT1 structures without disturbing these regions of the protein structure. Therefore, it remains possible that the inter-domain loop regions and C-termini of the eukaryotic NAT proteins are conformationally flexible, in order to accommodate substrate or inhibitor binding.

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