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Aromatic amino acid decarboxylase (AADC) deficiency is a rare autosomal neurometabolic disorder caused by a deficit of AADC, a pyridoxal 5'-phosphate (PLP)-dependent enzyme, which catalyzes the synthesis of dopamine and serotonin. While many studies have highlighted the molecular defects of the homozygous pathogenic variants, so far only a study investigated heterozygous variants at protein level. Here, we report a clinical case of one AADC deficiency compound heterozygous patient bearing the A91V mutation and the novel C410G mutation. To elucidate its enzymatic phenotype, the A91V and C410G homodimers were first expressed in Escherichia coli, purified and characterized. Although both apo variants display an unaltered overall tertiary structure, they show a ̴ 20-fold decreased PLP binding affinity. The C410G mutation only causes a ̴ 4-fold decrease of the catalytic efficiency, while the A91V mutation causes a 1300-fold decrease of the k cat /K m , and changes in the holoAADC consisting in a marked alteration of the tertiary structure and the coenzyme microenvironment. Structural analyses of these mutations are in agreement with these data. Unfortunately, the C410G/A91V heterodimer was constructed, expressed and purified in rather modest amount. Anyway, measurements of decarboxylase activity indicate that its putative k cat value is lower than that predicted by averaging the k cat values of the two parental enzymes. This indicates a negative interallelic complementation between the C410G and A91V monomers. Overall, this study allowed to relate the clinical to the enzymatic phenotype of the patient and to extend knowledge in the clinical and molecular pathogenesis of AADC deficiency., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Primary Hyperoxaluria Type I (PH1) is a disorder of glyoxylate metabolism caused by mutations in the human AGXT gene encoding liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP) dependent enzyme. Previous investigations highlighted that, although PH1 is characterized by a significant variability in terms of enzymatic phenotype, the majority of the pathogenic variants are believed to share both structural and functional defects, as mainly revealed by data on AGT activity and expression level in crude cellular extracts. However, the knowledge of the defects of the AGT variants at a protein level is still poor. We therefore performed a side-by-side comparison between normal AGT and nine purified recombinant pathogenic variants in terms of catalytic activity, coenzyme binding mode and affinity, spectroscopic features, oligomerization, and thermal stability of both the holo- and apo-forms. Notably, we chose four variants in which the mutated residues are located in the large domain of AGT either within the active site and interacting with the coenzyme or in its proximity, and five variants in which the mutated residues are distant from the active site either in the large or in the small domain. Overall, this integrated analysis of enzymatic activity, spectroscopic and stability information is used to (i) reassess previous data obtained with crude cellular extracts, (ii) establish which form(s) (i.e. holoenzyme and/or apoenzyme) and region(s) (i.e. active site microenvironment, large and/or small domain) of the protein are affected by each mutation, and (iii) suggest the possible therapeutic approach for patients bearing the examined mutations., (Copyright © 2011 Elsevier Inc. All rights reserved.)