Biochemical investigation of pathogenic missense mutations of human 4-amino butyrate aminotransferase towards the understanding of the molecular pathogenesis of GABA transaminase deficiency.

Gamma-amino butyrate aminotransferase (GABA-AT or ABAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of GABA and α-ketoglutarate into succinic semialdehyde and L-glutamate. In humans, the primary physiological role of GABA-AT is to control the level of GABA in neuronal tissues. Mutations on ABAT gene are associated to GABA-AT deficiency, an ultra-rare autosomal recessive disorder characterized by accelerated linear growth, severe psychomotor retardation, seizures, hypotonia, and hyperreflexia. So far, several missense pathogenic mutations of GABA-AT have been identified; however, their molecular effects at protein level have been poorly investigated. In this work a biochemical characterization of 10 pathogenic variants of human GABA-AT was carried out by expressing the protein in HEK-293 cells. Moreover, in-silico analyses of the variants were performed to corroborate the experimental findings. Altogether, the data obtained on protein expression level, GABA transaminase activity, and the predicted structural impact allowed us to classify the variants into three distinct groups, such as: (i) variants with strong structural and catalytic defects (p.P152S, p.L211F, and p.L478P); (ii) variants characterized mainly by a strong catalytic defect (p.R220K, p.Q296H, and p.R377W); (iii) variants exhibiting moderate structural and catalytic defects maintaining substantial transaminase activity (p.R92Q, p.F213C, p.G465D, and p.G465R). Based on these results, we provide a picture of the molecular defects of different GABA-AT pathogenic variants with the aim of gaining insights into the enzymatic phenotypes in GABA-AT deficiency.

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