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Amino acid N-acyltransferases in Human Metabolism

  • The human enzymes GLYAT (glycine N-acyltransferase), GLYATL1 (glutamine N-phenylacetyltransferase) and GLYATL2 (glycine N-acyltransferase-like protein 2) are not only important in the detoxification of xenobiotics via the human liver, but are also involved in the elimination of acyl residues that accumulate in the form of their coenzyme A (coA) esters in some rare inborn errors of metabolism. This concerns, for example, disorders in the degradation of branched-chain amino acids, such as isovaleric acidemia or propionic acidemia. In addition, they also assist in the elimination of ammonium, which is produced during the transamination of amino acids and accumulates in urea cycle defects. Sequence variants of the enzymes have also been investigated, which may provide evidence of impaired enzyme activities, from which therapy adjustments can potentially be derived. A modified Escherichia coli strain was chosen for the overexpression and partial biochemical characterization of the enzymes, which may allow solubility and proper folding. Since post-translational protein modifications are very limited in bacteria, we also attempted to overexpress the enzymes in HEK293 cells (human-derived). In addition to characterization via immunoblots and activity assays, intracellular localization of the enzymes was also performed using GFP coupling and confocal laser scanning microscopy in transfected HEK293 cells. The GLYATL2 enzyme may have tasks beyond detoxification and metabolic defects and the preliminary molecular biology work has been performed as part of this project - the enzyme activity determinations were outsourced to a co-supervised bachelor thesis. The enzyme activity determinations with purified recombinant human enzyme from Escherichia coli provided a threefold higher activity of the sequence variant p.(Asn156Ser) for GLYAT, which should be considered as the probably authentic wild type of the enzyme. In addition, a reduced activity of the GLYAT variant p.(Gln61Leu), which is very common in South Africa, was shown, which could be of particular importance in the treatment of isovaleric acidemia, which is also common in South Africa. Intracellularly, GLYAT and GLYATL1 could be localized mitochondrially. As the analyses have shown, sequence variations of GLYAT and GLYATL1 influence their enzyme activity. As an example, the GLYAT variant p.(Gln61Leu) is frequently found in South Africa. In the case of reduced GLYAT activity, patients could be increasingly treated with L-carnitine in the sense of an individualized therapy, since the conjugation of the toxic isovaleryl-coA with glycine is restricted by the GLYAT sequence variation. Activity-reducing variants identified in this project are of particular interest, as they may influence the treatment of certain metabolic defects.

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Document Type:Doctoral Thesis
Author:Daniel Schulke
Number of pages:217
Referee:Jörn Oliver Sass, Michael Gütschow
Publisher:Universitäts- und Landesbibliothek Bonn
Date of exam:2022/11/03
Contributing Corporation:Rheinische Friedrich-Wilhelms-Universität Bonn; Hochschule Bonn-Rhein-Sieg
Date of first publication:2022/12/22
Keyword:Benzoyl-coenzym A; Glutamin N-phenylacetyltransferase; Glycin N-acyltransferase; Glycine N-acyltransferase; Glycine conjugation; Glyzinkonjugation; Harnstoffzyklusdefekt; Isovalerianazidämie; Isovaleric acidemia; Phase II Reaktion; Phenylacetyl-coenzym A; benzoyl-coA; glutamine N-phenylacetyltransferase; phase II reaction; phenylacetyl-coA; urea cycle defect
Departments, institutes and facilities:Fachbereich Angewandte Naturwissenschaften
Dewey Decimal Classification (DDC):5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Entry in this database:2023/01/04