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

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.

Frequent sequence variants of human glycine N-acyltransferase (GLYAT) and inborn errors of metabolism

“…Distribution of sequence variation allowed characterization of four different alleles in addition to the reference allele. Due to the suspicious high allele frequency of c.467A>G, this variant may have been regarded as the real GLYAT wild-type [101], which has been corroborated by enormous high enzyme activity of p.(Asn156Ser) variant [129,152,154]. thioredoxin and glutaredoxin system that reduce disulfide bonds in bacteria cytoplasm, thus destroying tertiary protein structure [124].…”

“…Those, in combination with co-expressed GroES/GroEL [17], the Trx-tag fusion [33] and the compatible pET32 plasmid system [174] cover the certain compulsory requirements for the overexpression of eukaryotic GLYAT in a prokaryotic system. It has been used several times for the overexpression of GLYAT [129,152,154]. On the other side, mutations within these genes resulted in a decreased growing rate of E. coli Origami 2(DE3) in contrast to other E. coli strains [22,124] -a problematic challenge within the project, which will be described more precisely (Section 4.4.).…”

“…[107,152]. In a more recent publication, the glycine concentration was varied from 1 -200 mmol/L [129]. However, the choice of 200 mmol/L glycine represents the maximum concentration published in the literature and a multiple of the KMapp (glycine) of GLYAT [107,129].…”

“…In a more recent publication, the glycine concentration was varied from 1 -200 mmol/L [129]. However, the choice of 200 mmol/L glycine represents the maximum concentration published in the literature and a multiple of the KMapp (glycine) of GLYAT [107,129]. The maximum concentration of benzoyl-coA (200 µmol/L) was chosen due to literature data that confirm the usage of <200 µmol/L as maximum concentration for GLYAT activity assay [107,129,152,154].…”

Structure of glycine N-acyltransferase clarifies its catalytic mechanism

“…Due to its physiological relevance, human GLYAT has recently received considerable attention. Many groups have reported genetic polymorphisms in the GLYAT gene, and in vitro studies of the prevalent variants showed some of the mutations affect enzymatic activity [8][9][10][11] (Supplementary Table 2). Without a representative GLYAT structure, previous analyses were based on homology models generated using low identity templates (<12 %) 8 , which could introduce severe bias.…”