Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose
| Autor: | David J. Carrier, Frederica L. Theodoulou, Ronald J.A. Wanders, Hans R. Waterham, Alison Baker, Carlo W.T. van Roermund, Theresia A. Schaedler, Hong Lin Rong, Lodewijk IJlst, Stephen A. Baldwin |
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| Přispěvatelé: | Laboratory Genetic Metabolic Diseases, AGEM - Inborn errors of metabolism, APH - Methodology, Amsterdam Reproduction & Development (AR&D) |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Models Molecular Protein Conformation ATPase Arabidopsis Mutation Missense lcsh:Medicine ATP-binding cassette transporter Saccharomyces cerevisiae Spodoptera Article Cell Line 03 medical and health sciences Structure-Activity Relationship 0302 clinical medicine Adenosine Triphosphate Thioesterase Catalytic Domain Catalytic triad Membrane proteins Peroxisomes Animals Plant transporters lcsh:Science Adenosine Triphosphatases Multidisciplinary biology Chemistry Arabidopsis Proteins lcsh:R Mutagenesis Walker motifs Wild type Recombinant Proteins Transmembrane domain 030104 developmental biology Biochemistry Enzyme mechanisms biology.protein Mutagenesis Site-Directed lcsh:Q ATP-Binding Cassette Transporters Thiolester Hydrolases Fatty Acid Synthases Hydrophobic and Hydrophilic Interactions Oxidation-Reduction 030217 neurology & neurosurgery Oleic Acid Protein Binding |
| Zdroj: | Scientific Reports Scientific Reports, Vol 9, Iss 1, Pp 1-13 (2019) Scientific reports, 9(1):10502. Nature Publishing Group |
| ISSN: | 2045-2322 |
| Popis: | The peroxisomal ABC transporter, Comatose (CTS), a full length transporter from Arabidopsis has intrinsic acyl-CoA thioesterase (ACOT) activity, important for physiological function. We used molecular modelling, mutagenesis and biochemical analysis to identify amino acid residues important for ACOT activity. D863, Q864 and T867 lie within transmembrane helix 9. These residues are orientated such that they might plausibly contribute to a catalytic triad similar to type II Hotdog fold thioesterases. When expressed in Saccharomyces cerevisiae, mutation of these residues to alanine resulted in defective of β-oxidation. All CTS mutants were expressed and targeted to peroxisomes and retained substrate-stimulated ATPase activity. When expressed in insect cell membranes, Q864A and S810N had similar ATPase activity to wild type but greatly reduced ACOT activity, whereas the Walker A mutant K487A had greatly reduced ATPase and no ATP-dependent ACOT activity. In wild type CTS, ATPase but not ACOT was stimulated by non-cleavable C14 ether-CoA. ACOT activity was stimulated by ATP but not by non-hydrolysable AMPPNP. Thus, ACOT activity depends on functional ATPase activity but not vice versa, and these two activities can be separated by mutagenesis. Whether D863, Q864 and T867 have a catalytic role or play a more indirect role in NBD-TMD communication is discussed. |
| Databáze: | OpenAIRE |
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