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Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA

The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various substrates in this pathway and their competition for the pathway enzymes are currently unknown. The pathway consists of a mitochon...

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Autores principales: Rohwer, Johann M., Schutte, Chantelle, van der Sluis, Rencia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003330/
https://www.ncbi.nlm.nih.gov/pubmed/33803916
http://dx.doi.org/10.3390/ijms22063129
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author Rohwer, Johann M.
Schutte, Chantelle
van der Sluis, Rencia
author_facet Rohwer, Johann M.
Schutte, Chantelle
van der Sluis, Rencia
author_sort Rohwer, Johann M.
collection PubMed
description The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various substrates in this pathway and their competition for the pathway enzymes are currently unknown. The pathway consists of a mitochondrial xenobiotic/medium-chain fatty acid: coenzyme A (CoA) ligase (ACSM2B) and glycine N-acyltransferase (GLYAT). The catalytic mechanism and substrate specificity of both of these enzymes have not been thoroughly characterised. In this study, the level of evolutionary conservation of GLYAT missense variants and haplotypes were analysed. From these data, haplotype variants were selected (156Asn > Ser, [17Ser > Thr,156Asn > Ser] and [156Asn > Ser,199Arg > Cys]) in order to characterise the kinetic mechanism of the enzyme over a wide range of substrate concentrations. The 156Asn > Ser haplotype has the highest frequency and the highest relative enzyme activity in all populations studied, and hence was used as the reference in this study. Cooperative substrate binding was observed, and the kinetic data were fitted to a two-substrate Hill equation. The coding region of the GLYAT gene was found to be highly conserved and the rare 156Asn > Ser,199Arg > Cys variant negatively affected the relative enzyme activity. Even though the 156Asn > Ser,199Arg > Cys variant had a higher affinity for benzoyl-CoA (s(0.5,benz) = 61.2 µM), k(cat) was reduced to 9.8% of the most abundant haplotype 156Asn > Ser (s(0.5,benz) = 96.6 µM), while the activity of 17Ser > Thr,156Asn > Ser (s(0.5,benz) = 118 µM) was 73% of 156Asn > Ser. The in vitro kinetic analyses of the effect of the 156Asn > Ser,199Arg > Cys variant on human GLYAT enzyme activity indicated that individuals with this haplotype might have a decreased ability to metabolise benzoate when compared to individuals with the 156Asn > Ser variant. Furthermore, the accumulation of acyl-CoA intermediates can inhibit ACSM2B leading to a reduction in mitochondrial energy production.
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spelling pubmed-80033302021-03-28 Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA Rohwer, Johann M. Schutte, Chantelle van der Sluis, Rencia Int J Mol Sci Article The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various substrates in this pathway and their competition for the pathway enzymes are currently unknown. The pathway consists of a mitochondrial xenobiotic/medium-chain fatty acid: coenzyme A (CoA) ligase (ACSM2B) and glycine N-acyltransferase (GLYAT). The catalytic mechanism and substrate specificity of both of these enzymes have not been thoroughly characterised. In this study, the level of evolutionary conservation of GLYAT missense variants and haplotypes were analysed. From these data, haplotype variants were selected (156Asn > Ser, [17Ser > Thr,156Asn > Ser] and [156Asn > Ser,199Arg > Cys]) in order to characterise the kinetic mechanism of the enzyme over a wide range of substrate concentrations. The 156Asn > Ser haplotype has the highest frequency and the highest relative enzyme activity in all populations studied, and hence was used as the reference in this study. Cooperative substrate binding was observed, and the kinetic data were fitted to a two-substrate Hill equation. The coding region of the GLYAT gene was found to be highly conserved and the rare 156Asn > Ser,199Arg > Cys variant negatively affected the relative enzyme activity. Even though the 156Asn > Ser,199Arg > Cys variant had a higher affinity for benzoyl-CoA (s(0.5,benz) = 61.2 µM), k(cat) was reduced to 9.8% of the most abundant haplotype 156Asn > Ser (s(0.5,benz) = 96.6 µM), while the activity of 17Ser > Thr,156Asn > Ser (s(0.5,benz) = 118 µM) was 73% of 156Asn > Ser. The in vitro kinetic analyses of the effect of the 156Asn > Ser,199Arg > Cys variant on human GLYAT enzyme activity indicated that individuals with this haplotype might have a decreased ability to metabolise benzoate when compared to individuals with the 156Asn > Ser variant. Furthermore, the accumulation of acyl-CoA intermediates can inhibit ACSM2B leading to a reduction in mitochondrial energy production. MDPI 2021-03-18 /pmc/articles/PMC8003330/ /pubmed/33803916 http://dx.doi.org/10.3390/ijms22063129 Text en © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Rohwer, Johann M.
Schutte, Chantelle
van der Sluis, Rencia
Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA
title Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA
title_full Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA
title_fullStr Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA
title_full_unstemmed Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA
title_short Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA
title_sort functional characterisation of three glycine n-acyltransferase variants and the effect on glycine conjugation to benzoyl–coa
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003330/
https://www.ncbi.nlm.nih.gov/pubmed/33803916
http://dx.doi.org/10.3390/ijms22063129
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