Molecular heterogeneity in AADC deficiency: Variant-dependent effects on AADC activity

Aromatic L-amino acid decarboxylase (AADC), encoded by DDC gene, catalyzes the final step in dopamine and serotonin biosynthesis. Pathogenic DDC variants cause AADC deficiency, a severe neurometabolic disorder. This study aimed to elucidate previously uninvestigated DDC variant combinations: p.Pro47Ala/p.Arg447Cys and p.Cys261Phe/p.Gly354Ser, with respect to structural and functional characteristics. Recombinant heterodimeric and homodimeric AADC proteins were expressed in Escherichia coli and purified. Enzymatic activity was quantified via ion-pair reversed-phase HPLC with photodiode detection, while structural changes were analyzed using circular dichroism and molecular modelling. Functional characterization revealed that p.Pro47Ala, p.Gly354Ser, and p.Arg447Cys homodimers showed marked loss of activity towards both L-DOPA (88%, 97% and >99%, respectively) and L-5-hydroxytryptophan (88%, 99%, >99%, respectively), whereas p.Cys261Phe retained substantial activity (82% for L-DOPA and 75% for L-5-hydroxytryptophan). Kinetic analysis indicated a mild to severe decrease in substrate affinity for all homodimers, particularly for p.Arg447Cys. Furthermore, heterodimeric combinations showed positive complementation effects on decarboxylase reaction for both heterodimers. Surprisingly, p.Cys261Phe/p.Gly354Ser also restored the wild-type activity with L-DOPA and regained 43% of L-5-hydroxytryptophan activity, whereas p.Pro47Ala/p.Arg447Cys was 15% and 10% active towards L-DOPA and L-5-hydroxytryptophan with respect to the wild-type. Our results indicate that the pathogenicity of AADC variants depends on structural combination, complementation effects in heterodimeric species, and residual activity of homodimers, highlighting the complexity of polypeptide chain interactions in compound heterozygotes.