The establishment of a model system in which variants of the medium-chain ligase (ACSM) and glycine N-acyltransferase (GLYAT) can be co-expressed and analysed

Abstract

Even though glycine conjugation pathway discovery dates back to the 18th century, it is still an important detoxification pathway that is severely neglected in research. Xenobiotics are omnipresent in the environment (as pollutants) and are mainly used as food preservatives (e.g., sodium benzoate). The biphasic glycine detoxification pathway is important for detoxifying these xenobiotics by conjugation to glycine and then excretion into the urine. The first phase of the glycine conjugation pathway activates the xenobiotic to form a xenobiotic-CoA molecule in the presence of ATP, whereas the second phase conjugates glycine to the activated xenobiotic-CoA and releases CoA as a result. The hydrophilicity of xenobiotics is increased by conjugation with glycine to be excreted in the urine. Phase I activation of the glycine conjugation pathway is performed by ACSM2B (medium-chain fatty acid ligase, E.C. 2.6.1.2), and phase II conjugation is performed by GLYAT (glycine N-acyltransferase, E.C. 2.3.1.13). An additional feature of the glycine conjugation pathway is specifically activating medium-chain fatty acids to be used in β-oxidation as an additional energy-producing pathway. However, other substrates, such as organic acids (e.g., isovaleryl-CoA), could accumulate due to metabolic defects. This accumulation of organic acids may also enter the second phase of the glycine conjugation pathway to be conjugated and excreted. Previous studies on ACSM2B or GLYAT only focused on one variant, and some studies have already indicated detrimental effects in animal models and associated increased ingestion of xenobiotics with ADHD in children. These studies, however, did not focus on defective enzymes of the glycine conjugation pathway; therefore, research on the effect of genetic variation in the enzymes of the glycine conjugation pathway is still lacking. An expression model in which variants of ACSM2B and GLYAT can be expressed is, therefore, still a necessity in research of the glycine conjugation pathway. This establishment will also contribute to generating an enzymatic model of the glycine conjugation pathway. This study attempted to establish an expression model in which variants of ACSM2B, GLYAT, and GLYATL1 (one of the GLYAT paralogues) can be expressed and analysed. Two expression systems were analysed. The first was the coexpression system using the pETDuet-1_ACSM2B/GLYAT construct, and the second was the expression of pHis17_GLYAT and pET23a(+)_GLYATL1. Both systems used the OverExpress™ C41(DE3)pLysS expression host to express the highest haplotype of each gene. In the first expression system, soluble GLYAT expression was observed, and in the second expression system, soluble GLYAT and GLYATL1 expression were observed. ACSM2B could not be expressed, possibly due to its toxic effect on the expression host. The enzymes in the second expression system were purified, concentrated, and stored in Tris-HCl (pH 8.0) buffer after optimisation of the expression conditions. The matrices were analysed for the soluble lysate and purified, concentrated GLYAT and GLYATL1 before GC–MS analysis. Different enzyme reactions were set up using the C41(DE3)pLysS soluble lysate. The purified, concentrated GLYAT and GLYATL1 could not be used to set up enzyme reactions due to severe peak distortions. The substrates for each reaction varied between benzoyl-CoA, phenylacetyl-CoA, isovaleryl-CoA, glycine, and glutamine. Some of the reactions contained mixtures of the substrates. The reactions were then prepared for GC–MS analysis and run on an established GC–MS system. The GC–MS system measured the decrease (if possible) of the substrates (such as benzoyl-CoA) and the consequent increase of the end-products (such as hippuric acid). The extracted ion chromatograms indicated that the expressed soluble GLYAT (in combination with glycine) could effectively convert benzoyl-CoA to hippuric acid in both expression systems. The first system was only used to detect glycine conjugation to benzoyl-CoA. This is because it was the only xenobiotic substrate available at that point in the experimental phase. The soluble GLYAT in the second system could, however, convert some phenylacetyl-CoA in the presence of glycine, but no conversion was seen of phenylacetyl-CoA by GLYAT in the presence of glutamine. The expressed soluble GLYATL1 was not able to convert benzoyl-CoA. Soluble GLYATL1 was, however, able to successfully convert phenylacetyl-CoA in the presence of either glycine or glutamine. Isovaleryl-CoA and its products could not be measured, but the addition of isovaleryl-CoA to the GLYAT reaction, which contained benzoyl-CoA, indicated an inhibitory effect on the elimination of benzoyl-CoA. The overall results agreed with reported literature that two distinctive acyltransferase enzymes exist to detoxify benzoate (GLYAT + glycine) and phenylacetyl-CoA (GLYATL1 + glycine/glutamine). Furthermore, the results indicated that two successful expression systems were set up for GLYAT and one for GLYATL1, in which variants of these genes may be expressed and analysed. Further characterisation of all the enzymes involved in the glycine conjugation pathway and their variants still needs to be determined. This will contribute to the effect of genetic variation on the glycine conjugation pathway and lead to a more characterised glycine conjugation pathway to establish an enzymatic model of this biphasic pathway. The more characterised glycine conjugation becomes a better understanding of the glycine conjugation pathway and its role in human health. Furthermore, it will lead to better treatments for individuals who suffer from metabolic defects (such as isovaleric acidemia) or disorders (such as ADHD in children).