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

Johann M. Rohwer,Chantelle Schutte,Rencia van der Sluis

doi:10.3390/ijms22063129

Johann M. Rohwer, Chantelle Schutte + Show 1 more

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https://doi.org/10.3390/ijms22063129

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Abstract

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 (s0.5,benz = 61.2 µM), kcat was reduced to 9.8% of the most abundant haplotype 156Asn > Ser (s0.5,benz = 96.6 µM), while the activity of 17Ser > Thr,156Asn > Ser (s0.5,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.

Highlights

The glycine conjugation pathway is a two-step enzymatic reaction responsible for the metabolism/detoxification of natural substrates from (i) food (for example., salicylate [1], dietary polyphenols and medium-chain fatty acids (MCFAs)), (ii) xenobiotics, and (iii) metabolites produced from organic acidaemia [2,3,4] (Figure 1)
In the case of glycine conjugation, it has been shown that glycine N-acyltransferase (GLYAT) expression is transcriptionally down-regulated in hepatocellular carcinoma specimens [28]
Glycine conjugation can be influenced by several factors, including the availability of ATP, coenzyme A (CoA), and glycine, variants in the ACSM2B and GLYAT genes, and differential expression of ACSM2B and GLYAT

Summary

Introduction

The glycine conjugation pathway is a two-step enzymatic reaction responsible for the metabolism/detoxification of natural substrates from (i) food (for example., salicylate [1], dietary polyphenols and medium-chain fatty acids (MCFAs)), (ii) xenobiotics (for example benzoate), and (iii) metabolites produced from organic acidaemia [2,3,4] (Figure 1). Benzoate and salicylate are activated to an acyl-coenzyme A (CoA) by the mitochondrial xenobiotic/medium-chain fatty acid: CoA ligases (ACSM2B,EC 6.2.1.2) [5,6] and subsequently conjugated to glycine by glycine N-acyltransferase (GLYAT, EC 2.3.1.13) to form hippuric acid and salicyluric acid, respectively [3,7,8]. Benzoic acid consumption has been linked to adverse effects such as diarrhoea, metabolic acidosis, tremors, and childhood hyperactivity syndrome [19,20] These symptoms might be the result of low levels of glycine as glycine conjugation is needed to detoxify the high levels of benzoate. Studies have shown that benzoic acid inhibits the elimination of salicyluric acid, and that the accumulation of salicyl-CoA may result in toxicity and liver damage [23,24]

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