Glycine-N-acyltransferase (GLYAT, EC 2.3.1.13) has been characterised in a number of species including: humans, chimpanzees, rhesus monkeys and bovines. The characterisation of GLYAT from various species contributes to a better understanding of the diversity of the enzyme which in turn might help improve the current understanding of detoxification in mammals. The GLYAT enzyme of both the chacma baboon and vervet monkey has not been characterised. In this project, tissue samples were obtained from a chacma baboon (Papio ursinus) and a vervet monkey (Chlorocebus pygerythrus) to determine the nucleic acid sequence that encodes GLYAT in these two species to broaden our current understanding on the diversity of GLYAT in primates. A liver of a chacma baboon was used to extract total RNA. Complementary DNA (cDNA) was synthesised using an oligo (dT) primer. An open reading frame (ORF) encoding GLYAT of the chacma baboon was amplified with a PCR (polymerase chain reaction) using primers designed from a human GLYAT transcript. The PCR product containing an ORF encoding GLYAT of the chacma baboon was cloned, sequenced and expressed. The recombinant GLYAT of the chacma baboon expressed well in bacteria, but was insoluble and did not have enzyme activity. A crude cytoplasmic extract was prepared from the liver of a chacma baboon. The objective was to compare enzyme activity between the native and recombinant GLYAT. The prepared liver extract from the chacma baboon was assayed for enzyme activity and compared to the activity in a liver extract from bovine, previously prepared by Ms M Snyders. Both the chacma baboon and bovine liver extracts had GLYAT enzyme activity. To obtain sequence information on vervet monkey GLYAT, leukocytes were isolated from blood obtained from a living vervet monkey. A human GLYAT gene sequence was used as a reference DNA sequence in the design of PCR primers that were used to amplify the exons of GLYAT of the vervet monkey. All six GLYAT exons were individually amplified and PCR products were sequenced. The sequences were combined to reconstruct an ORF encoding GLYAT of the vervet monkey. The ORFs coding the GLYAT of both chacma baboon and vervet monkey were found to be 888 bp long (excluding stop codon) and encoded a protein of 296 amino acids. A fragment of 1256 bp of the chacma baboon GLYAT transcript was sequenced. The two GLYAT ORF sequences were translated to amino acid sequences and aligned to that of GLYAT of primates obtained from the Ensembl sequence database. The GLYAT amino acid sequences of the chacma baboon, vervet monkey and rhesus monkey formed a related group, distinct from other primates. The chacma baboon and vervet monkey sequences were 99 % identical to the rhesus monkey sequence and 92.6 % identical to the human sequence. There were 4 new variations introduced by GLYAT amino acid sequences from the chacma baboon and the vervet monkey. The vervet monkey introduced an isoleucine in place of a valine at position 32 and an arginine in place of a histidine or glutamine at position 224. The chacma baboon introduced a tyrosine in place of isoleucine at position 201 and an arginine in place of histidine or glutamine at position 240. The knowledge generated in this project will broaden the understanding of GLYAT diversity relating to GLYAT in primates. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011

Glycine-N-acyltransferase (GLYAT, EC 2.3.1.13) has been characterised in a number of species including: humans, chimpanzees, rhesus monkeys and bovines. The characterisation of GLYAT from various species contributes to a better understanding of the diversity of the enzyme which in turn might help improve the current understanding of detoxification in mammals. The GLYAT enzyme of both the chacma baboon and vervet monkey has not been characterised. In this project, tissue samples were obtained from a chacma baboon (Papio ursinus) and a vervet monkey (Chlorocebus pygerythrus) to determine the nucleic acid sequence that encodes GLYAT in these two species to broaden our current understanding on the diversity of GLYAT in primates. A liver of a chacma baboon was used to extract total RNA. Complementary DNA (cDNA) was synthesised using an oligo (dT) primer. An open reading frame (ORF) encoding GLYAT of the chacma baboon was amplified with a PCR (polymerase chain reaction) using primers designed from a human GLYAT transcript. The PCR product containing an ORF encoding GLYAT of the chacma baboon was cloned, sequenced and expressed. The recombinant GLYAT of the chacma baboon expressed well in bacteria, but was insoluble and did not have enzyme activity. A crude cytoplasmic extract was prepared from the liver of a chacma baboon. The objective was to compare enzyme activity between the native and recombinant GLYAT. The prepared liver extract from the chacma baboon was assayed for enzyme activity and compared to the activity in a liver extract from bovine, previously prepared by Ms M Snyders. Both the chacma baboon and bovine liver extracts had GLYAT enzyme activity. To obtain sequence information on vervet monkey GLYAT, leukocytes were isolated from blood obtained from a living vervet monkey. A human GLYAT gene sequence was used as a reference DNA sequence in the design of PCR primers that were used to amplify the exons of GLYAT of the vervet monkey. All six GLYAT exons were individually amplified and PCR products were sequenced. The sequences were combined to reconstruct an ORF encoding GLYAT of the vervet monkey. The ORFs coding the GLYAT of both chacma baboon and vervet monkey were found to be 888 bp long (excluding stop codon) and encoded a protein of 296 amino acids. A fragment of 1256 bp of the chacma baboon GLYAT transcript was sequenced. The two GLYAT ORF sequences were translated to amino acid sequences and aligned to that of GLYAT of primates obtained from the Ensembl sequence database. The GLYAT amino acid sequences of the chacma baboon, vervet monkey and rhesus monkey formed a related group, distinct from other primates. The chacma baboon and vervet monkey sequences were 99 % identical to the rhesus monkey sequence and 92.6 % identical to the human sequence. There were 4 new variations introduced by GLYAT amino acid sequences from the chacma baboon and the vervet monkey. The vervet monkey introduced an isoleucine in place of a valine at position 32 and an arginine in place of a histidine or glutamine at position 224. The chacma baboon introduced a tyrosine in place of isoleucine at position 201 and an arginine in place of histidine or glutamine at position 240. The knowledge generated in this project will broaden the understanding of GLYAT diversity relating to GLYAT in primates. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011

Thorough investigation of the glycine conjugation pathway has been neglected over the last 30 years. Environmental factors, nutrition, and the chronic use of medications are increasing the exposure of humans to benzoate and drugs that are metabolized to acyl-CoA intermediates. Glycine conjugation of mitochondrial acyl-CoAs, catalysed 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 characterised in detail. Therefore, one of the objectives of this thesis was to develop a better understanding of glycine conjugation and its role in metabolism. In humans and animals a number of endogenous and xenobiotic organic acids are conjugated to glycine. 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, recently it was proposed that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilised as neurotransmitters in the central nervous systems of animals. The glycine deportation hypothesis was based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. A thorough review of the literature for this thesis showed that the major role of glycine conjugation, however, is to dispose of the end products of phenylpropionate metabolism. The review also introduced the new perspective that mitochondrial glycine conjugation prevents the accumulation of benzoate in the mitochondrial matrix by forming hippuric acid a less lipophilic conjugate that can be more readily transported out of the mitochondria. Although organic anion transporters can export benzoate from the matrix, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not the transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. Lastly, glycine conjugation of benzoate also 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. To date, no defect of the glycine conjugation pathway has been reported and this, together with the fact that GLYAT plays an important role in hepatic metabolism, suggests that this pathway is essential for survival. 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. Significant interindividual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. The main aim of this thesis was to investigate and characterise the genetic variation in the coding region of the GLYAT gene. This was accomplished by firstly, investigating the influence of non-synonymous single nucleotide polymorphisms (SNPs) on the enzyme activity of a recombinant human GLYAT and secondly, by analysing the level of genetic variation in the coding region of the GLYAT gene using existing worldwide population data. To investigate the influence of non-synonymous SNPs in the GLYAT gene on the enzyme activity, a recombinant human GLYAT was prepared, and characterised. Site-directed mutagenesis was used to generate six 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. The results showed that SNP variations in the human GLYAT gene can influence the kinetic properties of the enzyme. The genetic variation data of the human GLYAT open reading frame (ORF) available on public databases was investigated by formulating the hypothesis that due to the essential nature of the glycine conjugation pathway, the genetic variation in the ORF of the GLYAT gene should be low and that deleterious alleles will be found at low frequencies. Data from the i) 1000 Genome Project, ii) the HapMap Project, and iii) the Khoi-San/Bantu Sequencing Project was downloaded from available databases. Sequence data of the coding region of a small cohort of South African Afrikaner Caucasian individuals was also generated and included in the analyses. In the GLYAT ORF of the 1537 individuals analysed, only two haplotypes (S156 and T17S156) out of 14 haplotypes were identified in all populations as having the highest haplotype frequencies (70% and 20% respectively). The S156C199 and S156H131 haplotypes, which have a deleterious effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. The results of this study indicated that the GLYAT ORF is remarkably conserved, which supports the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. The findings presented in this thesis highlight the importance that future investigations should determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2015

Thorough investigation of the glycine conjugation pathway has been neglected over the last 30 years. Environmental factors, nutrition, and the chronic use of medications are increasing the exposure of humans to benzoate and drugs that are metabolized to acyl-CoA intermediates. Glycine conjugation of mitochondrial acyl-CoAs, catalysed 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 characterised in detail. Therefore, one of the objectives of this thesis was to develop a better understanding of glycine conjugation and its role in metabolism. In humans and animals a number of endogenous and xenobiotic organic acids are conjugated to glycine. 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, recently it was proposed that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilised as neurotransmitters in the central nervous systems of animals. The glycine deportation hypothesis was based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. A thorough review of the literature for this thesis showed that the major role of glycine conjugation, however, is to dispose of the end products of phenylpropionate metabolism. The review also introduced the new perspective that mitochondrial glycine conjugation prevents the accumulation of benzoate in the mitochondrial matrix by forming hippuric acid a less lipophilic conjugate that can be more readily transported out of the mitochondria. Although organic anion transporters can export benzoate from the matrix, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not the transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. Lastly, glycine conjugation of benzoate also 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. To date, no defect of the glycine conjugation pathway has been reported and this, together with the fact that GLYAT plays an important role in hepatic metabolism, suggests that this pathway is essential for survival. 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. Significant interindividual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. The main aim of this thesis was to investigate and characterise the genetic variation in the coding region of the GLYAT gene. This was accomplished by firstly, investigating the influence of non-synonymous single nucleotide polymorphisms (SNPs) on the enzyme activity of a recombinant human GLYAT and secondly, by analysing the level of genetic variation in the coding region of the GLYAT gene using existing worldwide population data. To investigate the influence of non-synonymous SNPs in the GLYAT gene on the enzyme activity, a recombinant human GLYAT was prepared, and characterised. Site-directed mutagenesis was used to generate six 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. The results showed that SNP variations in the human GLYAT gene can influence the kinetic properties of the enzyme. The genetic variation data of the human GLYAT open reading frame (ORF) available on public databases was investigated by formulating the hypothesis that due to the essential nature of the glycine conjugation pathway, the genetic variation in the ORF of the GLYAT gene should be low and that deleterious alleles will be found at low frequencies. Data from the i) 1000 Genome Project, ii) the HapMap Project, and iii) the Khoi-San/Bantu Sequencing Project was downloaded from available databases. Sequence data of the coding region of a small cohort of South African Afrikaner Caucasian individuals was also generated and included in the analyses. In the GLYAT ORF of the 1537 individuals analysed, only two haplotypes (S156 and T17S156) out of 14 haplotypes were identified in all populations as having the highest haplotype frequencies (70% and 20% respectively). The S156C199 and S156H131 haplotypes, which have a deleterious effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. The results of this study indicated that the GLYAT ORF is remarkably conserved, which supports the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. The findings presented in this thesis highlight the importance that future investigations should determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2015

Humans are continuously challenged by harmful endogenous and xenobiotic substances. Detoxification is the ability to neutralise and remove these substances from the body. Glycine N-acyltransferase, EC 2.3.1.13 (GLYAT) is a key enzyme in detoxification. GLYAT catalyses an amino acid (glycine) conjugation reaction in phase II of detoxification. It is expected that, similar to what has been observed in the Cytochrome P450 enzymes, variations within the GLYAT gene may lead to altered enzyme activity that may affect the efficacy of detoxification. The aim of this study was to identify genetic variations within the GLYAT gene of a cohort of individuals whose GLYAT activity has been biochemically characterized. Biochemical profiles of phase I and II detoxification of a number of individuals was screened to select those with possible aberrant GLYAT activity. Eighteen selected individuals agreed to participate in the study. The 23.21 kb GLYAT gene of the participants was amplified in four fragments and sent for pyrosequencing (Roche GS FLX titanium) at Inqaba Biotec. The results were analysed with the Lasergene software package from DNAStar (Madison, Wisconsin, USA). A total of 94 variations were identified from the Next Generation Sequencing data. Of these three found in the exons were known variations and four variations located in the exons were novel. A total of 62 known and 25 novel variations were identified in the introns of the GLYAT gene. Sanger sequencing verified 70.29% (68 in total) of the variation, which included 12 novel variations, of which one is located in exon six. Real-time quantitative PCR (qPCR) experiments were conducted and the data analysed using CopyCaller software to identify copy number variations within the cohort. It was found that participant 17 may have multiple copies of parts of the 3-terminal end of the gene (exons five and six), which might have an effect on GLYAT activity. Variations could possibly affect GLYAT activity, but the data was inconclusive and must be confirmed. Some of the variations could possibly affect GLYAT activity, but no correlation could be made between the variations identified during this study and the cohort’s detoxification ability. Further studies needs to be conducted to establish the effect of the variations in combination with one another on GLYAT activity. If some of these variations affect GLYAT activity such data might shed some light on variations observed between the glycine conjugation ability of individuals. Such information could eventually be of value in treatment of inborn errors of metabolism. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Humans are continuously challenged by harmful endogenous and xenobiotic substances. Detoxification is the ability to neutralise and remove these substances from the body. Glycine N-acyltransferase, EC 2.3.1.13 (GLYAT) is a key enzyme in detoxification. GLYAT catalyses an amino acid (glycine) conjugation reaction in phase II of detoxification. It is expected that, similar to what has been observed in the Cytochrome P450 enzymes, variations within the GLYAT gene may lead to altered enzyme activity that may affect the efficacy of detoxification. The aim of this study was to identify genetic variations within the GLYAT gene of a cohort of individuals whose GLYAT activity has been biochemically characterized. Biochemical profiles of phase I and II detoxification of a number of individuals was screened to select those with possible aberrant GLYAT activity. Eighteen selected individuals agreed to participate in the study. The 23.21 kb GLYAT gene of the participants was amplified in four fragments and sent for pyrosequencing (Roche GS FLX titanium) at Inqaba Biotec. The results were analysed with the Lasergene software package from DNAStar (Madison, Wisconsin, USA). A total of 94 variations were identified from the Next Generation Sequencing data. Of these three found in the exons were known variations and four variations located in the exons were novel. A total of 62 known and 25 novel variations were identified in the introns of the GLYAT gene. Sanger sequencing verified 70.29% (68 in total) of the variation, which included 12 novel variations, of which one is located in exon six. Real-time quantitative PCR (qPCR) experiments were conducted and the data analysed using CopyCaller software to identify copy number variations within the cohort. It was found that participant 17 may have multiple copies of parts of the 3-terminal end of the gene (exons five and six), which might have an effect on GLYAT activity. Variations could possibly affect GLYAT activity, but the data was inconclusive and must be confirmed. Some of the variations could possibly affect GLYAT activity, but no correlation could be made between the variations identified during this study and the cohort’s detoxification ability. Further studies needs to be conducted to establish the effect of the variations in combination with one another on GLYAT activity. If some of these variations affect GLYAT activity such data might shed some light on variations observed between the glycine conjugation ability of individuals. Such information could eventually be of value in treatment of inborn errors of metabolism. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Afin de s'adapter à son environnement chimique, l'organisme a développé au cours de l'évolution des systèmes enzymatiques capables de transformer de nombreuses molécules étrangères ou xénobiotiques (médicaments, composés toxiques, carcinogènes...), le plus souvent de nature hydrophobe, en métabolites suffisamment hydrophiles pour être plus facilement excrétés par voie urinaire et/ou biliaire. Certaines de ces enzymes sont également impliquées dans des processus cataboliques ou de biosynthèses de composés endogènes (acides gras, rétinoïdes, stéroïdes, prostaglandines…). Ces enzymes jouent ainsi un rôle fondamental à la fois dans la défense de l'organisme face à son environnement chimique et dans des processus physiologiques essentiels. On comprend dès lors que s'il existe, chez certains individus, des anomalies de séquence ou de structure des gènes codant pour ces enzymes, une partie de la population présentera une susceptibilité particulière à certaines molécules de l'environnement, voire des dysfonctionnements de certaines réactions biologiques indispensables. Les travaux de cette thèse s'inscrivent dans cette démarche. Dans un premier temps, ils ont consisté à évaluer la nature et l'étendue de la variabilité de la séquence nucléotidique de trois gènes codants pour les enzymes CYP4A11, CYP4A22 et la Glycine N-acyltransférase (GLYAT). Dans un deuxième temps, les analyses fonctionnelles des variations de séquence identifiées ont été abordées par des approches in silico et in vitro. Les cytochromes P450 CYP4A11 et CYP4A22, participent à la biotransformation de composés endogènes et sont impliqués plus particulièrement dans la voie d’activation de l’acide arachidonique. Des travaux récents suggèrent que des anomalies génétiques de ces enzymes constituent des facteurs de susceptibilité à l’hypertension artérielle chez l’homme. Nous avons ainsi analysé les variations de séquence du gène CYP4A11 et CYP4A22 dans des échantillons d'ADN provenant de volontaires sains. Au total, 26 polymorphismes ont été identifiés et 5 nouveaux CYP4A* allèles ont été caractérisés pour chaque isoforme CYP4A. Les structures 3D des protéines CYP4A ont été construites et validées pour l’analyse de l’impact des mutations identifiées. Bien que des travaux supplémentaires soient nécessaires pour confirmer le lien entre le polymorphisme génétique du CYP4A11 et du CYP4A22 et l’hypertension artérielle, ce travail représente la première description et caractérisation du polymorphisme génétique des isoformes CYP4A dans une population Française. De plus, nous avons mise en évidence une variabilité interethnique de ce polymorphisme génétique dans différentes populations testées. La glycine N-acyltransférase ou GLYAT est une enzyme impliquée dans la détoxication de xénobiotiques contenant un groupement carboxylique par conjugaison d’un résidu de glycine. Sept variations de séquence de la GLYAT ont été identifiées et quatre nouveaux GLYAT* allèles ont été caractérisés. La localisation des certaines mutations dans des structures secondaires très conservées de la protéine suggère un impact sur l’activité catalytique de cette enzyme. Bien que les conséquences cliniques potentielles de ces variations restent encore à étudier, ces résultats seront utiles pour de futures études d’association de ce polymorphisme génétique de la GLYAT avec les altérations de détoxications de xénobiotiques contenant un groupement carboxylique comme l’aspirine, certains pesticides ou le toluène. / Through evolution, in order to adapt to its chemical environment, the human organism has developed enzymatic systems that can transform exogenous molecules or xenobiotic (drugs, toxins, carcinogens…), generally of hydrophobic nature, in metabolites more easily excretable via urinary or biliary tract. Some of these enzymes are also involved in catabolic processes or in the biosynthesis of endogenous compounds (fatty acids, retinoids, steroids, prostaglandins…). These enzymes thus play a major role in the protective response of the body toward chemicals and in essential physiological processes. The existence of anomalies in the sequence or structure of the genes encoding these enzymes can expose carriers of these anomalies to particular susceptibility toward xenobiotics or to impairment of essential biological reactions. In a first step, we investigated the nature and extent of the sequence variability of three genes coding for the enzymes CYP4A11, CYP4A22 and Glycine N-acyltransferase (GLYAT). In a second step, functional analyses of sequence variations were carried out, by in silico and in vitro experiments. The CYP4A11 and CYP4A22 genes are the only members of the human CYP4A subfamily. The activity of the recently identified CYP4A22 isoform is still unknown, but the CYP4A11 isoform is know as a ω-hydroxylase of the arachidonic acid, which converted into 20-hydroxyeicosatetraenoic acid (20-HETE). Several studies have shown that genetic anomalies of CYP4A are likely to contribute for susceptibility to hypertension in humans. We analyzed the sequence variations of the CYP4A11 and CYP4A22 genes in genomic DNA samples of healthy volunteers. A total of 26 polymorphisms were identified and 5 novel CYP4A* alleles were characterized for each CYP4A gene. The CYP4A 3D models were built and validated to analyse the potential impact of sequence variations identified. This work represents the first description and characterisation of genetic polymorphism of the human CYP4A genes in a French population. The glycine N-acyltranferase or GLYAT plays an important role in the detoxification of xenobiotics containing a carboxylic group via conjugation with a glycine residue. Seven sequence variations of the GLYAT gene were identified and four novel GLYAT* alleles were characterized. Localisation of missense mutations in predicted secondary structures suggest that these variants might have a potential role on the GLYAT protein activity. These results could be helpful in investigating the potential association of GLYAT variants with an incidence of reduced efficiency in xenobiotic carboxylic acids detoxification in humans, such as acetylsalicylic acid, pesticides, and solvents (Toluene).

Afin de s'adapter à son environnement chimique, l'organisme a développé au cours de l'évolution des systèmes enzymatiques capables de transformer de nombreuses molécules étrangères ou xénobiotiques (médicaments, composés toxiques, carcinogènes...), le plus souvent de nature hydrophobe, en métabolites suffisamment hydrophiles pour être plus facilement excrétés par voie urinaire et/ou biliaire. Certaines de ces enzymes sont également impliquées dans des processus cataboliques ou de biosynthèses de composés endogènes (acides gras, rétinoïdes, stéroïdes, prostaglandines...). Ces enzymes jouent ainsi un rôle fondamental à la fois dans la défense de l'organisme face à son environnement chimique et dans des processus physiologiques essentiels. On comprend dès lors que s'il existe, chez certains individus, des anomalies de séquence ou de structure des gènes codant pour ces enzymes, une partie de la population présentera une susceptibilité particulière à certaines molécules de l'environnement, voire des dysfonctionnements de certaines réactions biologiques indispensables. Les travaux de cette thèse s'inscrivent dans cette démarche. Dans un premier temps, ils ont consisté à évaluer la nature et l'étendue de la variabilité de la séquence nucléotidique de trois gènes codants pour les enzymes CYP4A11, CYP4A22 et la Glycine N-acyltransférase (GLYAT). Dans un deuxième temps, les analyses fonctionnelles des variations de séquence identifiées ont été abordées par des approches in silico et in vitro. Les cytochromes P450 CYP4A11 et CYP4A22, participent à la biotransformation de composés endogènes et sont impliqués plus particulièrement dans la voie d'activation de l'acide arachidonique. Des travaux récents suggèrent que des anomalies génétiques de ces enzymes constituent des facteurs de susceptibilité à l'hypertension artérielle chez l'homme. Nous avons ainsi analysé les variations de séquence du gène CYP4A11 et CYP4A22 dans des échantillons d'ADN provenant de volontaires sains. Au total, 26 polymorphismes ont été identifiés et 5 nouveaux CYP4A* allèles ont été caractérisés pour chaque isoforme CYP4A. Les structures 3D des protéines CYP4A ont été construites et validées pour l'analyse de l'impact des mutations identifiées. Bien que des travaux supplémentaires soient nécessaires pour confirmer le lien entre le polymorphisme génétique du CYP4A11 et du CYP4A22 et l'hypertension artérielle, ce travail représente la première description et caractérisation du polymorphisme génétique des isoformes CYP4A dans une population Française. De plus, nous avons mise en évidence une variabilité interethnique de ce polymorphisme génétique dans différentes populations testées. La glycine N-acyltransférase ou GLYAT est une enzyme impliquée dans la détoxication de xénobiotiques contenant un groupement carboxylique par conjugaison d'un résidu de glycine. Sept variations de séquence de la GLYAT ont été identifiées et quatre nouveaux GLYAT* allèles ont été caractérisés. La localisation des certaines mutations dans des structures secondaires très conservées de la protéine suggère un impact sur l'activité catalytique de cette enzyme. Bien que les conséquences cliniques potentielles de ces variations restent encore à étudier, ces résultats seront utiles pour de futures études d'association de ce polymorphisme génétique de la GLYAT avec les altérations de détoxications de xénobiotiques contenant un groupement carboxylique comme l'aspirine, certains pesticides ou le toluène.

Despite being the first biochemical reaction to be discovered, the glycine conjugation pathway remains poorly characterised. It has generally been assumed that glycine conjugation serves to increase the water solubility of organic acids, such as benzoic acid and isovaleric acid, in order to facilitate urinary excretion of these compounds. However, it was recently suggested that the conjugation of glycine to benzoate should be viewed as a neuroregulatory process that prevents the accumulation of glycine, a neurotransmitter, to toxic levels. The true importance of glycine conjugation in metabolism is therefore not well understood. However, no genetic defect of glycine conjugation has ever been reported. This seems to suggest that glycine conjugation is a fundamentally important metabolic process, whatever its function may be. Therefore, a major objective of this thesis was to develop a deeper understanding of glycine conjugation and its metabolic significance. A review of the literature on GLYAT and glycine conjugation suggested that the primary purpose of glycine conjugation is indeed to detoxify benzoate and other aromatic acids of dietary origin. However, the commonly held assumption, that glycine conjugation increases the water solubility of aromatic acids in order to facilitate urinary excretion, seems to be incorrect. A better explanation for the detoxification of benzoate by means of glycine conjugation is based on hydrophilicity, not water solubility. Because of its lipophilic nature, benzoic acid is capable of passively diffusing across the mitochondrial inner membrane into the matrix space, where it accumulates due to the pH gradient over the inner membrane. Although benzoate can be exported from the matrix by organic anion transporters, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. The conversion of benzoate to hippurate is a two-step process. First, benzoate is activated by an ATP-dependent acid:CoA ligase (ACSM2A) to form the more reactive benzoyl-CoA. Second, glycine N-acyltransferase (GLYAT) catalyses the formation of hippurate and CoASH from benzoyl-CoA and glycine. Another major objective of this thesis was to gain a better understanding of the structure and function of the GLYAT enzyme. While the substrate selectivity and enzyme kinetics of GLYAT have been investigated to some extent, almost nothing has been published on the structure, active site, or catalytic mechanism of GLYAT. Furthermore, while interindividual variation in the rate of glycine conjugation has been reported by several researchers, it is not known if, or how, genetic variation in the human GLYAT gene contributes to this interindividual variation. To address these issues, systems for the bacterial expression of recombinant bovine GLYAT and recombinant human GLYAT were developed. Because no crystal structure of GLYAT has been reported, homology modelling was used to generate a molecular model of bovine GLYAT. By comparing the molecular model to other acyltransferases for which the catalytic residues were known, Glu227 of bovine GLYAT was identified as a potential catalytic residue. Site directed mutagenesis was used to generate an E227Q mutant recombinant bovine GLYAT lacking the proposed catalytic residue. Characterisation of this mutant suggested that Glu227 was indeed the catalytic residue, and the GLYAT catalytic mechanism was elucidated. The molecular model was also used to identify Asn131 of bovine GLYAT as a potential active site residue. Site-directed mutagenesis was used to generate an N131C mutant, which was sensitive to inhibition by the sulfhydryl reagent DTNB. This suggests that the Asn131 residue of bovine GLYAT may be situated in the active site of bovine GLYAT, but more work is needed to confirm this result. Finally, site-directed mutagenesis was used to generate variants of recombinant human GLYAT corresponding to six of the known SNPs in the human GLYAT gene. Expression and characterisation of the recombinant human GLYAT variants revealed that the enzyme activity and KM (benzoyl-CoA) parameter of the recombinant human GLYAT were influenced by SNPs in the human GLYAT gene. This suggests that genetic variation in the human GLYAT gene could partly explain the interindividual variation in the rate of glycine conjugation observed in humans. Interestingly, the SNPs that negatively influenced enzyme activity also had low allele frequencies, suggesting that there may be some selective advantage to having high GLYAT activity. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

Awareness of detoxification, nowadays known as biotransformation, has become an integral part of our daily lives. It is a modern buzz word that is used to promote anything from health food to enhancement of performance in sports. Another lesser known application for detoxification is as a therapy for alleviating symptoms of inborn errors of metabolism. Detoxification is the process where endogenous and xenobiotic metabolites are transformed to less harmful products, in the liver and kidneys, in two phases. Phase 1 detoxification includes oxidation, hydroxylation, dehydrogenation metabolic reduction and hydrolysis. Phase 2 detoxification uses conjugation reactions to increase hydrophillicty of metabolites for excretion in bile and urine. Glycine N-acyltransferse (GLYAT; EC 2.3.1.13) is one of the amino acid conjugation enzymes. There are two variants of human GLYAT. I focused on the full-length mRNA human GLYAT isoform a, with a long term view of using it as a viable therapeutic enzyme for enhanced detoxification of harmful metabolites. I investigated if it is possible to clone and express a biologically active GLYAT. To achieve this goal I used three expression systems: traditional bacterial expression using the pET system; second generation cold shock bacterial expression using the pCOLDTF expression vector to improve solubility of the recombinant protein; and baculovirus expression in insect cells since therein some form of post translation glycosylation of the recombinant protein can occur which might improve solubility and ensure biological activity. The recombinant GLYAT expressed well in all three expression systems but was aggregated and no enzyme activity could be detected. A denature and renature system was also used to collect aggregated recombinant GLYAT and used to try to refold the recombinant protein in appropriate refolding buffers to improve solubility and obtain biological activity. The solubility of the recombinant GLYAT was improved but it remained biologically inactive. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.