Rencia van der Sluis

A new perspective on the importance of glycine conjugation in the metabolism of aromatic acids

Abstract A number of endogenous and xenobiotic organic acids are conjugated to glycine, in animals ranging from mosquitoes to humans. Glycine conjugation has generally been assumed to be a detoxification mechanism, increasing the water solubility of organic acids in order to facilitate urinary excretion. However, the recently proposed glycine deportation hypothesis states that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilized as neurotransmitters in the central nervous systems of animals. This hypothesis is based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. In this review it will be argued that the major role of glycine conjugation is to dispose of the end products of phenylpropionate metabolism. Furthermore, glucuronidation, which occurs in the endoplasmic reticulum, would not be ideal for the detoxification of free benzoate, which has been shown to accumulate in the mitochondrial matrix. Glycine conjugation, however, prevents accumulation of benzoic acid in the mitochondrial matrix by forming hippurate, a less lipophilic conjugate that can be more readily transported out of the mitochondria. Finally, it will be explained that the glycine conjugation of benzoate, a commonly used preservative, exacerbates the dietary deficiency of glycine in humans. Because the resulting shortage of glycine can negatively influence brain neurochemistry and the synthesis of collagen, nucleic acids, porphyrins, and other important metabolites, the risks of using benzoate as a preservative should not be underestimated.

Glycine conjugation: importance in metabolism, the role of glycine N-acyltransferase, and factors that influence interindividual variation

Introduction: Glycine conjugation of mitochondrial acyl-CoAs, catalyzed by glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13), is an important metabolic pathway responsible for maintaining adequate levels of free coenzyme A (CoASH). However, because of the small number of pharmaceutical drugs that are conjugated to glycine, the pathway has not yet been characterized in detail. Here, we review the causes and possible consequences of interindividual variation in the glycine conjugation pathway. Areas covered: The authors review the importance of CoASH in metabolism, formation and toxicity of xenobiotic acyl-CoAs, and mechanisms for restoring levels of CoASH. They focus on GLYAT, glycine conjugation, how genetic variation in the GLYAT gene could influence glycine conjugation, and the emerging roles of glycine metabolism in cancer and musculoskeletal development. Expert opinion: The substrate selectivity of GLYAT and its variants needs to be further characterized, as organic acids can be toxic if the corresponding acyl-CoA is not a substrate for glycine conjugation. GLYAT activity affects mitochondrial ATP production, glycine availability, CoASH availability, and the toxicity of various organic acids. Therefore, variation in the glycine conjugation pathway could influence liver cancer, musculoskeletal development, and mitochondrial energy metabolism.

Characterisation of the influence of genetic variations on the enzyme activity of a recombinant human glycine N-acyltransferase

Human glycine N-acyltransferase (human GLYAT) detoxifies a wide range of endogenous and xenobiotic metabolites, including benzoate and salicylate. Significant inter-individual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. To investigate the influence of single nucleotide polymorphisms (SNPs) in the GLYAT coding sequence on enzyme activity, we expressed and characterised a recombinant human GLYAT. Site-directed mutagenesis was used to generate six non-synonymous SNP variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. We also generated an E227Q mutant, which lacks the catalytic residue proposed by Badenhorst et al. (2012). This mutant was inactive compared to the wild-type recombinant human GLYAT. A molecular model of human GLYAT containing coenzyme A (CoA) was generated which revealed that the inactivity of the R199C variant could be due to the substitution of the highly conserved Arg(199) and destabilisation of an α-loop-α motif which is important for substrate binding in the GNAT superfamily. The finding that SNP variations in the human GLYAT gene influence the kinetic properties of the enzyme may explain some of the inter-individual variation in glycine conjugation capacity, which is relevant to the metabolism of xenobiotics such as aspirin and the industrial solvent xylene, and to the treatment of some metabolic disorders.

Conservation of the coding regions of the glycine N-acyltransferase gene further suggests that glycine conjugation is an essential detoxification pathway

Thorough investigation of the glycine conjugation pathway has been neglected. No defect of the glycine conjugation pathway has been reported and this could reflect the essential role of glycine conjugation in hepatic metabolism. Therefore, we hypothesised that genetic variation in the open reading frame (ORF) of the GLYAT gene should be low and that deleterious alleles would be found at low frequencies. This hypothesis was investigated by analysing the genetic variation of the human GLYAT ORF using data available in public databases. We also sequenced the GLYAT ORF of a small cohort of South African Afrikaner Caucasian individuals. In total, data from 1537 individuals was analysed. The two most prominent GLYAT haplotypes in all populations analysed, were S156 (70%) and T17S156 (20%). The S156C199 and S156H131 haplotypes, which have a negative effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. In the Afrikaner Caucasian cohort a novel Q61L SNP occurring at a high frequency (12%) was detected. The results of this study indicated that the GLYAT ORF is highly conserved and supported the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. These findings emphasise the importance of future investigations to determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics.

The use of p-aminobenzoic acid as a probe substance for the targeted profiling of glycine conjugation

Glycine conjugation facilitates the metabolism of toxic aromatic acids, capable of disrupting mitochondrial integrity. Owing to the high exposure to toxic substrates, characterization of individual glycine conjugation capacity, and its regulatory factors has become increasingly important. Aspirin and benzoate have been employed for this purpose; however, adverse reactions, aspirin intolerance, and Reyes syndrome in children are substantial drawbacks. The goal of this study was to investigate p‐aminobenzoic acid (PABA) as an alternative glycine conjugation probe. Ten human volunteers participated in a PABA challenge test, and p‐aminohippuric acid (PAHA), p‐acetamidobenzoic acid, and p‐acetamidohippuric acid were quantified in urine. The glycine N‐acyltransferase gene of the volunteers was also screened for two polymorphisms associated with normal and increased enzyme activity. All of the individuals were homozygous for increased enzyme activity, but excretion of PAHA varied significantly (16–56%, hippurate ratio). The intricacies of PABA metabolism revealed possible limiting factors and the potential of PABA as an indicator of Phase 0 biotransformation.

Xenobiotic/medium chain fatty acid: CoA ligase – a critical review on its role in fatty acid metabolism and the detoxification of benzoic acid and aspirin

ABSTRACT Introduction: Activation of fatty acids by the acyl-CoA synthetases (ACSs) is the vital first step in fatty acid metabolism. The enzymatic and physiological characterization of the human xenobiotic/medium chain fatty acid: CoA ligases (ACSMs) has been severely neglected even though xenobiotics, such as benzoate and salicylate, are detoxified through this pathway. Areas covered: This review will focus on the nomenclature and substrate specificity of the human ACSM ligases; the biochemical and enzymatic characterization of ACSM1 and ACSM2B; the high sequence homology of the ACSM2 genes (ACSM2A and ACSM2B) as well as what is currently known regarding disease association studies. Expert opinion: Several discrepancies exist in the current literature that should be taken note of. For example, the single nucleotide polymorphisms (SNPs) reported to be associated with aspirin metabolism and multiple risk factors of metabolic syndrome are incorrect. Kinetic data on the substrate specificity of the human ACSM ligases are non-existent and currently no data exist on the influence of SNPs on the enzyme activity of these ligases. One of the biggest obstacles currently in the field is that glycine conjugation is continuously studied as a one-step process, which means that key regulatory factors of the two individual steps remain unknown.

Analyses of the genetic diversity and protein expression variation of the acyl: CoA medium-chain ligases, ACSM2A and ACSM2B

Benzoate (found in milk and widely used as preservative), salicylate (present in fruits and the active component of aspirin), dietary polyphenols produced by gut microbiota, metabolites from organic acidemias, and medium-chain fatty acids (MCFAs) are all metabolised/detoxified by the glycine conjugation pathway. Xenobiotics are first activated to an acyl-CoA by the mitochondrial xenobiotic/medium-chain fatty acid: CoA ligases (ACSMs) and subsequently conjugated to glycine by glycine N-acyltransferase (GLYAT). The MCFAs are activated to acyl-CoA by the ACSMs before entering mitochondrial β-oxidation. This two-step enzymatic pathway has, however, not been thoroughly investigated and the biggest gap in the literature remains the fact that studies continuously characterise the pathway as a one-step reaction. There are no studies available on the interaction/competition of the various substrates involved in the pathway, whilst very little research has been done on the ACSM ligases. To identify variants/haplotypes that should be characterised in future detoxification association studies, this study assessed the naturally observed sequence diversity and protein expression variation of ACSM2A and ACSM2B. The allelic variation, haplotype diversity, Tajima’s D values, and phylogenetic analyses indicated that ACSM2A and ACSM2B are highly conserved. This confirmed an earlier hypothesis that the glycine conjugation pathway is highly conserved and essential for life as it maintains the CoA and glycine homeostasis in the liver mitochondria. The protein expression analyses showed that ACSM2A is the predominant transcript in liver. Future studies should investigate the effect of the variants identified in this study on the substrate specificity of these proteins.

Draft de novo genome sequence of Agapornis roseicollis for application in avian breeding

ABSTRACT In aviculture, lovebirds are considered one of the most popular birds to keep. This African parakeet is known for its range of plumage colors and ease to tame. Plumage variation is the most important price-determining trait of these birds, and also the main selection criterion for breeders. Currently, no genetic screening tests for traits of economic importance or to confirm pedigree data are available for any of the nine lovebird species. As a starting point to develop these tests, the de novo genome of Agapornis roseicollis (rosy-faced lovebird) was sequenced, assembled, and annotated. Sequencing was done on the Illumina HiSeq 2000 platform and the assembly was performed using SOAPdenovo v2.04. The genome was found to be 1.1 Gb in size and 16,044 genes were identified and annotated. This compared well with other previously sequenced avian genomes, such as the chicken, zebra finch, and budgerigar. To assess genome completeness, the number of benchmarking universal single-copy orthologs were identified in the genome. This was compared to other previously assembled avian genomes and the results indicated that the genome will be useful in the development of genetic screening tests to aid lovebird breeders in selecting breeding pairs.

New insights into the catalytic mechanism of human glycine N-acyltransferase

Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi‐substrate kinetic parameters of a recombinant human glycine N‐acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC–ESI‐MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi–Bi and/or ping‐pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl‐CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis–Menten reaction mechanism.