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Title: Structural Basis of Substrate-Binding Specificity of Human Arylamine N-acetyltransferases

Abstract

The human arylamine N-acetyltransferases NAT1 and NAT2 play an important role in the biotransformation of a plethora of aromatic amine and hydrazine drugs. They are also able to participate in the bioactivation of several known carcinogens. Each of these enzymes is genetically variable in human populations, and polymorphisms in NAT genes have been associated with various cancers. Here we have solved the high resolution crystal structures of human NAT1 and NAT2, including NAT1 in complex with the irreversible inhibitor 2-bromoacetanilide, a NAT1 active site mutant, and NAT2 in complex with CoA, and have refined them to 1.7-, 1.8-, and 1.9- Angstroms resolution, respectively. The crystal structures reveal novel structural features unique to human NATs and provide insights into the structural basis of the substrate specificity and genetic polymorphism of these enzymes.

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
959567
Report Number(s):
BNL-82553-2009-JA
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US201016%%711
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 282; Journal Issue: 41
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; AMINES; AROMATICS; CARCINOGENS; CRYSTAL STRUCTURE; ENZYMES; GENES; GENETICS; HUMAN POPULATIONS; HYDRAZINE; RESOLUTION; SPECIFICITY; SUBSTRATES; national synchrotron light source

Citation Formats

Wu,H., Dombrovsky, L., Tempel, W., Martin, F., Loppnau, P., Goodfellow, G., Grant, D., and Plotnikov, A. Structural Basis of Substrate-Binding Specificity of Human Arylamine N-acetyltransferases. United States: N. p., 2007. Web. doi:10.1074/jbc.M704138200.
Wu,H., Dombrovsky, L., Tempel, W., Martin, F., Loppnau, P., Goodfellow, G., Grant, D., & Plotnikov, A. Structural Basis of Substrate-Binding Specificity of Human Arylamine N-acetyltransferases. United States. doi:10.1074/jbc.M704138200.
Wu,H., Dombrovsky, L., Tempel, W., Martin, F., Loppnau, P., Goodfellow, G., Grant, D., and Plotnikov, A. Mon . "Structural Basis of Substrate-Binding Specificity of Human Arylamine N-acetyltransferases". United States. doi:10.1074/jbc.M704138200.
@article{osti_959567,
title = {Structural Basis of Substrate-Binding Specificity of Human Arylamine N-acetyltransferases},
author = {Wu,H. and Dombrovsky, L. and Tempel, W. and Martin, F. and Loppnau, P. and Goodfellow, G. and Grant, D. and Plotnikov, A.},
abstractNote = {The human arylamine N-acetyltransferases NAT1 and NAT2 play an important role in the biotransformation of a plethora of aromatic amine and hydrazine drugs. They are also able to participate in the bioactivation of several known carcinogens. Each of these enzymes is genetically variable in human populations, and polymorphisms in NAT genes have been associated with various cancers. Here we have solved the high resolution crystal structures of human NAT1 and NAT2, including NAT1 in complex with the irreversible inhibitor 2-bromoacetanilide, a NAT1 active site mutant, and NAT2 in complex with CoA, and have refined them to 1.7-, 1.8-, and 1.9- Angstroms resolution, respectively. The crystal structures reveal novel structural features unique to human NATs and provide insights into the structural basis of the substrate specificity and genetic polymorphism of these enzymes.},
doi = {10.1074/jbc.M704138200},
journal = {Journal of Biological Chemistry},
number = 41,
volume = 282,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • A computational study was performed to better understand the differences between human arylamine N-acetyltransferase (NAT) 1 and 2. Homology models were constructed from available crystal structures and comparisons of the active site residues 125, 127, and 129 for these two enzymes provide insight into observed substrate differences. The NAT2 model provided a basis for understanding how some of the common mutations may affect the structure of the protein. Molecular dynamics simulations of the human NAT models and the template structure (NAT from Mycobacterium smegmatis) were performed and showed the models to be stable and reasonable. Docking studies of hydroxylated heterocyclicmore » amines in the models of NAT1 and NAT2 probed the differences exhibited by these two proteins with mutagenic agents. The hydroxylated heterocyclic amines were only able to fit into the NAT2 active site, and an alternative binding site by the P-loop was found using our models and will be discussed. Additionally, quantum mechanical calculations were performed to study the O-acetylation reaction of the hydroxylated heterocyclic amines N-OH MeIQx and N-OH PhIP. This study has given us insight into why there are substrate differences among isoenzymes and explains some of the polymorphic activity differences.« less
  • Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.
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