title: Oligomeric State and Thermal Stability of Apo- and Holo- Human Ornithine δ-Aminotransferase.
abstract: 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 Tmvalues 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, Tmvalues 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.
authors.affiliation: Department of Neuroscience, Biomedicine and Movement Sciences (Section of Biological Chemistry), University of Verona, Strada Le Grazie 8, 37134, Verona, Italy. riccardo.montioli@univr.it.
authors.forename: Riccardo
authors.identifier: 0000-0001-5906-1236
authors.initials: R
authors.lastname: Montioli
authors.affiliation: Department of Biochemical Sciences, University "La Sapienza", Rome, Italy.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Zamparelli
authors.affiliation: Department of Neuroscience, Biomedicine and Movement Sciences (Section of Biological Chemistry), University of Verona, Strada Le Grazie 8, 37134, Verona, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Borri Voltattorni
authors.affiliation: Department of Neuroscience, Biomedicine and Movement Sciences (Section of Biological Chemistry), University of Verona, Strada Le Grazie 8, 37134, Verona, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Cellini
mesh_terms: D019943:Amino Acid Substitution; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics; D006358:Hot Temperature; D006801:Humans; D020125:Mutation, Missense; D009953:Ornithine-Oxo-Acid Transaminase / Q000737:chemistry* / Q000235:genetics; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: D001051:Apoenzymes; D020035:Holoenzymes; D009953:Ornithine-Oxo-Acid Transaminase
keywords: Coenzyme; Interface contacts; Protein stability; Pyridoxal 5′-phosphate; Quaternary structure; Tetramer–dimer equilibrium