Biochemical investigation of the effects of pathogenic missense mutations on human ω-aminotransferases involved in rare genetic disorders

The ornithine aminotransferase (OAT) and the γ-aminobutyric acid aminotransferase (GABA-AT) are two ω-aminotransferases belonging to the fold type-I family of PLP-dependent enzymes. Despite the two enzymes present structural and catalytic similarities, they play very different metabolic roles. The OAT is involved in the control of the L-ornithine levels in all tissues, while the GABA-AT acts mainly in neurons, by controlling the level of GABA, a very important neurotransmitter. Both OAT and GABA-AT are linked to rare metabolic disorders. In particular, the deficiency of OAT leads to the onset of gyrate atrophy of the choroid and retina (GA), while a deficit of GABA-AT leads to the onset of GABA-transaminase deficiency. A crucial step in the understanding of the molecular pathogenesis beyond GA and GABA-AT deficiency, is the identification of the molecular defects of each pathogenic variant. By obtaining a specific genotype-phenotype associated with each patient it is possible to reduce the variability of response and gain more phenotype-targeted therapies. This research aims to understand how pathogenic missense mutations, identified in the OAT and GABA-AT sequences, affect the catalytic activity and/or the structural properties of the enzymes. Among the OAT pathogenic variants, we focused on those involving residues located at the monomer-monomer interface, a critical region for the enzyme maturation. The purified recombinant human OAT G51D, G121D, R154L, Y158S, T181M, and P199Q variants were investigated through spectroscopic, kinetic, and chromatographic analyses. Furthermore, unsolved aspects on the OAT oligomerization started a deeper investigation by photo-crosslinking through the incorporation of non-canonical amino acids. On the other hand, all the GABA-AT pathogenic variants, R92Q, P152S, L211F, R220K, Q296H, R377W, G465R, p.L478P, F213C, and G465D, were expressed in HEK293 cells and characterized in their catalytic activity and expression levels. According to the results we obtained, it has been possible to highlight the main molecular defects associated with the interface OAT pathogenic variants. Even if to a different extent, all the variants presented reduced thermal stability, increased exposure of hydrophobic surface, shifted the tetramer-dimer dissociation equilibrium towards the dimeric structure and reduced catalytic activity. In terms of catalytic defects, the Y158S, T181M, and P199Q presented consistent reduction of the catalytic efficiency. Instead, the G51D and G121D resulted to maintain a significant catalytic activity but are prone to lose the coenzyme during the catalytic steps, converting the enzyme into the inactive and unstable apo-form. Finally, the R154L resulted not able to catalyse the first half-reaction, thus impeding product formation. On the other side, the GABA-AT pathogenic variants were grouped and classified according to their expression level and relative specific activity. In particular, the first group includes the P152S, L211F, and L478P variants, which exhibit low expression levels and no residual catalytic activity; group two presents the Q296H, R377W and R220K variants, exhibiting high expression levels and a very low, or none, residual catalytic ability; and, finally, the third group contains the R92Q, F213C, G465D and G465R variants that, although they are defective in both expression and catalysis, globally displayed milder impact of the mutations with respect to the other groups of variants. All data collected allowed to draw a picture of the molecular defects of the pathogenic variants of OAT and GABA-AT. Moreover, they allowed to acquire information about the structure-function relationships of two enzyme that have a great biochemical and clinical relevance.