Aromatic L-Amino Acid Decarboxylase (AADC) is the enzyme responsible for the synthesis of two essential neurotransmitter dopamine and serotonin from L-Dopa and L-hydroxytryptophan. AADC owes its specific catalytic activity to the chemistry of its cofactor, pyrydoxal-5’-phosphate (PLP). Almost 20 years ago, the crystal structure of a mammalian holoAADC (porcine, sharing 90% of sequence identity) was solved and the availability of its 3D structure paved the way to structural studies. Moreover, 10 years later, human apoAADC structure was published, shedding light on the conformational rearrangement occurring on the apo enzyme upon addition of PLP. Importantly, apo and holoAADC structures provided crucial insights for the comprehension of the pathogenicity of a number of AADC deficiency associated variants. AADC deficiency (OMIM#608643) is a rare autosomal recessive inborn disease due to missense mutations in the AADC gene. Patients bearing these mutations show mild to severe phenotypes, whose destiny is often fatal. Due to the rarity of the disease and to the heterogeneous response to the treatments, medications are not often satisfactory. In the past years, some efforts on human recombinant AADC pathogenic variants have tried to provide support to the research on AADC deficiency by means of biochemical and biophysical approaches determining the impact of the amino acid substitutions on the enzyme features. Here, a further contribution to the comprehension of the AADC deficiency is provided. The crystal structure of human holoAADC has been solved under different conditions, both in its native and ligand bound form. The combination of crystallographic studies, molecular dynamics simulations (MD) and site directed mutagenesis uncovered novel aspects of the AADC structure-function relationship. Moreover, the characterization of 21 novel identified pathogenic variants (spread on each AADC domain, N-terminal, Large and C-terminal Domains) led to the widening of the range of enzymatic phenotypes associated to AADC deficiency. The proposed combination of biochemical and kinetic studies permitted to determine correlations between structural and functional signals. Enzymatic phenotypes span from variants characterized by a mild phenotypes to variants (mainly located at the NTD-CTD interface) whose dramatic structural defects lead to a catalytic incompetence. In addition, MD simulations and in solutions data point out a critical role for the loop3 element that contains the essential catalytic residue Tyr332. A group of variants affecting loop3 has been identified as catalytically incompetent and their structural features have been dissected thanks also to the solving of the crystal structure of pathogenic variant L353P, which constitutes the first solved structure of an AADC variant. Altogether, this study on human AADC provides new elements for the comprehension of the structure-function relationship of AADC with a particular focus on protein dynamics and mobility. Lastly, structural details might represent the basis for both the designing of novel specific inhibitors and for a better comprehension of the molecular aspects of the variants associated with the AADC deficiency.
L’enzima Decarbossilasi degli L-amino acidi aromatici (AADC) è responsabile della sintesi di due neurotrasmettitori essenziali: la dopamina e la serotonina. AADC deve la sua attività catalitica alla chimica del suo cofattore, il piridossale 5’-fosfato (PLP). La struttura cristallografica dell’enzima da mammifero (precisamente da maiale che ha il 90% di identità con l’enzima umano) nella sua forma olo venne risolta venti anni fa e tale risoluzione aprì la strada ad importanti studi strutturali. Dieci anni dopo venne pubblicata la struttura umana di AADC nella sua forma apo evidenziando quali cambiamenti conformazionali avvengono quando il PLP viene legato dall’enzima. Le strutture apo e olo AADC hanno avuto notevole importanza per la comprensione della patogenicità di varianti enzimatiche associate alla malattia chiamata ‘Deficit da AADC’ (AADCd, OMIM#608643). Questa malattia autosomica recessiva molto rara è dovuta prevalentemente a mutazioni missenso sul gene AADC. I pazienti affetti da AADCd mostrano un’am
|Titolo:||HUMAN AROMATIC L-AMINO ACID DECARBOXYLASE: WHEN STRUCTURE AND MOBILITY DRIVE EFFICIENT CATALYSIS. IMPLICATIONS FOR AADC DEFICIENCY|
BISELLO, GIOVANNI [Writing – Original Draft Preparation] (Corresponding)
|Data di pubblicazione:||2021|
|Appare nelle tipologie:||07.13 Doctoral Thesis|