Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding

BACKGROUND: The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous...

Descripción completa

Detalles Bibliográficos
Autores principales: Laurieri, Nicola, Kawamura, Akane, Westwood, Isaac M, Varney, Amy, Morris, Elizabeth, Russell, Angela J, Stanley, Lesley A, Sim, Edith
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258814/
https://www.ncbi.nlm.nih.gov/pubmed/25432241
http://dx.doi.org/10.1186/2050-6511-15-68
_version_ 1782347923805175808
author Laurieri, Nicola
Kawamura, Akane
Westwood, Isaac M
Varney, Amy
Morris, Elizabeth
Russell, Angela J
Stanley, Lesley A
Sim, Edith
author_facet Laurieri, Nicola
Kawamura, Akane
Westwood, Isaac M
Varney, Amy
Morris, Elizabeth
Russell, Angela J
Stanley, Lesley A
Sim, Edith
author_sort Laurieri, Nicola
collection PubMed
description BACKGROUND: The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut. METHODS: To determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards “(MOUSE)NAT2-specific” arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated. RESULTS: Comparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes. CONCLUSIONS: Three non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/2050-6511-15-68) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-4258814
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-42588142014-12-09 Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding Laurieri, Nicola Kawamura, Akane Westwood, Isaac M Varney, Amy Morris, Elizabeth Russell, Angela J Stanley, Lesley A Sim, Edith BMC Pharmacol Toxicol Research Article BACKGROUND: The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut. METHODS: To determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards “(MOUSE)NAT2-specific” arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated. RESULTS: Comparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes. CONCLUSIONS: Three non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/2050-6511-15-68) contains supplementary material, which is available to authorized users. BioMed Central 2014-11-29 /pmc/articles/PMC4258814/ /pubmed/25432241 http://dx.doi.org/10.1186/2050-6511-15-68 Text en © Laurieri et al.; licensee BioMed Central Ltd. 2014 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Laurieri, Nicola
Kawamura, Akane
Westwood, Isaac M
Varney, Amy
Morris, Elizabeth
Russell, Angela J
Stanley, Lesley A
Sim, Edith
Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding
title Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding
title_full Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding
title_fullStr Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding
title_full_unstemmed Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding
title_short Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding
title_sort differences between murine arylamine n-acetyltransferase type 1 and human arylamine n-acetyltransferase type 2 defined by substrate specificity and inhibitor binding
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258814/
https://www.ncbi.nlm.nih.gov/pubmed/25432241
http://dx.doi.org/10.1186/2050-6511-15-68
work_keys_str_mv AT laurierinicola differencesbetweenmurinearylaminenacetyltransferasetype1andhumanarylaminenacetyltransferasetype2definedbysubstratespecificityandinhibitorbinding
AT kawamuraakane differencesbetweenmurinearylaminenacetyltransferasetype1andhumanarylaminenacetyltransferasetype2definedbysubstratespecificityandinhibitorbinding
AT westwoodisaacm differencesbetweenmurinearylaminenacetyltransferasetype1andhumanarylaminenacetyltransferasetype2definedbysubstratespecificityandinhibitorbinding
AT varneyamy differencesbetweenmurinearylaminenacetyltransferasetype1andhumanarylaminenacetyltransferasetype2definedbysubstratespecificityandinhibitorbinding
AT morriselizabeth differencesbetweenmurinearylaminenacetyltransferasetype1andhumanarylaminenacetyltransferasetype2definedbysubstratespecificityandinhibitorbinding
AT russellangelaj differencesbetweenmurinearylaminenacetyltransferasetype1andhumanarylaminenacetyltransferasetype2definedbysubstratespecificityandinhibitorbinding
AT stanleylesleya differencesbetweenmurinearylaminenacetyltransferasetype1andhumanarylaminenacetyltransferasetype2definedbysubstratespecificityandinhibitorbinding
AT simedith differencesbetweenmurinearylaminenacetyltransferasetype1andhumanarylaminenacetyltransferasetype2definedbysubstratespecificityandinhibitorbinding