Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening

Akane Kawamura; James Graham; Adeel Mushtaq; Stefanos A. Tsiftsoglou; Gregory M. Vath; Patrick E. Hanna; Carston R. Wagner; Edith Sim

doi:10.1016/j.bcp.2004.09.014

Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening

Akane Kawamura, James Graham, Adeel Mushtaq, Stefanos A. Tsiftsoglou, Gregory M. Vath, Patrick E. Hanna, Carston R. Wagner, Edith Sim

Research output: Contribution to journal › Article › peer-review

93 Scopus citations

Abstract

Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

Original language	English (US)
Pages (from-to)	347-359
Number of pages	13
Journal	Biochemical Pharmacology
Volume	69
Issue number	2
DOIs	https://doi.org/10.1016/j.bcp.2004.09.014
State	Published - Jan 15 2005

Bibliographical note

Funding Information:
We thank Haiqing Wang (University of Minnesota, USA), Dr. Edward Brooke and Isaac Westwood (University of Oxford, UK) for helpful discussions and advice. We are also grateful to the Wellcome Trust for financial support and the Oxford University Kobe foundation for a scholarship (A.K.). Syrian hamster liver samples were kindly donated by Olivia Hibbitt (University of Oxford, UK).

Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.

Keywords

AcetylCoA/CoA derivatives
Arylamine N-acetyltransferase
Arylamines
High-throughput screening
Recombinant protein
Xenobiotic metabolism

Publisher link

10.1016/j.bcp.2004.09.014

Find It

Find It at U of M Libraries

Cite this

@article{3a07f3874b5e4940872d2f56c377082e,

title = "Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening",

abstract = "Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.",

keywords = "AcetylCoA/CoA derivatives, Arylamine N-acetyltransferase, Arylamines, High-throughput screening, Recombinant protein, Xenobiotic metabolism",

author = "Akane Kawamura and James Graham and Adeel Mushtaq and Tsiftsoglou, {Stefanos A.} and Vath, {Gregory M.} and Hanna, {Patrick E.} and Wagner, {Carston R.} and Edith Sim",

note = "Funding Information: We thank Haiqing Wang (University of Minnesota, USA), Dr. Edward Brooke and Isaac Westwood (University of Oxford, UK) for helpful discussions and advice. We are also grateful to the Wellcome Trust for financial support and the Oxford University Kobe foundation for a scholarship (A.K.). Syrian hamster liver samples were kindly donated by Olivia Hibbitt (University of Oxford, UK). Copyright: Copyright 2017 Elsevier B.V., All rights reserved.",

year = "2005",

month = jan,

day = "15",

doi = "10.1016/j.bcp.2004.09.014",

language = "English (US)",

volume = "69",

pages = "347--359",

journal = "Biochemical Pharmacology",

issn = "0006-2952",

publisher = "Elsevier Inc.",

number = "2",

}

TY - JOUR

T1 - Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening

AU - Kawamura, Akane

AU - Graham, James

AU - Mushtaq, Adeel

AU - Tsiftsoglou, Stefanos A.

AU - Vath, Gregory M.

AU - Hanna, Patrick E.

AU - Wagner, Carston R.

AU - Sim, Edith

N1 - Funding Information: We thank Haiqing Wang (University of Minnesota, USA), Dr. Edward Brooke and Isaac Westwood (University of Oxford, UK) for helpful discussions and advice. We are also grateful to the Wellcome Trust for financial support and the Oxford University Kobe foundation for a scholarship (A.K.). Syrian hamster liver samples were kindly donated by Olivia Hibbitt (University of Oxford, UK). Copyright: Copyright 2017 Elsevier B.V., All rights reserved.

PY - 2005/1/15

Y1 - 2005/1/15

N2 - Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

AB - Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

KW - AcetylCoA/CoA derivatives

KW - Arylamine N-acetyltransferase

KW - Arylamines

KW - High-throughput screening

KW - Recombinant protein

KW - Xenobiotic metabolism

UR - http://www.scopus.com/inward/record.url?scp=13844276136&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=13844276136&partnerID=8YFLogxK

U2 - 10.1016/j.bcp.2004.09.014

DO - 10.1016/j.bcp.2004.09.014

M3 - Article

C2 - 15627487

AN - SCOPUS:13844276136

SN - 0006-2952

VL - 69

SP - 347

EP - 359

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

IS - 2

ER -

Eukaryotic arylamine N-acetyltransferase investigation of substrate specificity by high-throughput screening

Abstract

Bibliographical note

Keywords

Publisher link

Find It

Other files and links

Fingerprint

Cite this