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Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase

Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5′-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- an...

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Autores principales: Montioli, Riccardo, Zamparelli, Carlotta, Borri Voltattorni, Carla, Cellini, Barbara
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer US 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432616/
https://www.ncbi.nlm.nih.gov/pubmed/28345116
http://dx.doi.org/10.1007/s10930-017-9710-5
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author Montioli, Riccardo
Zamparelli, Carlotta
Borri Voltattorni, Carla
Cellini, Barbara
author_facet Montioli, Riccardo
Zamparelli, Carlotta
Borri Voltattorni, Carla
Cellini, Barbara
author_sort Montioli, Riccardo
collection PubMed
description Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5′-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent T(m) values of 46 and 67 °C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer–dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, T(m) values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10930-017-9710-5) contains supplementary material, which is available to authorized users.
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spelling pubmed-54326162017-05-31 Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase Montioli, Riccardo Zamparelli, Carlotta Borri Voltattorni, Carla Cellini, Barbara Protein J Article Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5′-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent T(m) values of 46 and 67 °C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer–dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, T(m) values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10930-017-9710-5) contains supplementary material, which is available to authorized users. Springer US 2017-03-27 2017 /pmc/articles/PMC5432616/ /pubmed/28345116 http://dx.doi.org/10.1007/s10930-017-9710-5 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Article
Montioli, Riccardo
Zamparelli, Carlotta
Borri Voltattorni, Carla
Cellini, Barbara
Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase
title Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase
title_full Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase
title_fullStr Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase
title_full_unstemmed Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase
title_short Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase
title_sort oligomeric state and thermal stability of apo- and holo- human ornithine δ-aminotransferase
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432616/
https://www.ncbi.nlm.nih.gov/pubmed/28345116
http://dx.doi.org/10.1007/s10930-017-9710-5
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