학술논문
Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine [delta]-Aminotransferase
Article
Article
Document Type
Academic Journal
Source
The Protein Journal. June 2017, Vol. 36 Issue 3, p174, 12 p.
Subject
Language
English
ISSN
1572-3887
Abstract
Author(s): Riccardo Montioli [sup.1], Carlotta Zamparelli [sup.2], Carla Borri Voltattorni [sup.1], Barbara Cellini [sup.1] Author Affiliations: (1) 0000 0004 1763 1124, grid.5611.3, Department of Neuroscience, Biomedicine and Movement Sciences (Section [...]
Human ornithine [delta]-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.sub.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.sub.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.
Human ornithine [delta]-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.sub.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.sub.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.