title: The novel R347g pathogenic mutation of aromatic amino acid decarboxylase provides additional molecular insights into enzyme catalysis and deficiency.
abstract: We report here a clinical case of a patient with a novel mutation (Arg347→Gly) in the gene encoding aromatic amino acid decarboxylase (AADC) that is associated with AADC deficiency. The variant R347G in the purified recombinant form exhibits, similarly to the pathogenic mutation R347Q previously studied, a 475-fold drop of kcat compared to the wild-type enzyme. In attempting to unravel the reason(s) for this catalytic defect, we have carried out bioinformatics analyses of the crystal structure of AADC-carbidopa complex with the modelled catalytic loop (residues 328-339). Arg347 appears to interact with Phe103, as well as with both Leu333 and Asp345. We have then prepared and characterized the artificial F103L, R347K and D345A mutants. F103L, D345A and R347K exhibit about 13-, 97-, and 345-fold kcat decrease compared to the wild-type AADC, respectively. However, unlike F103L, the R347G, R347K and R347Q mutants as well as the D345A variant appear to be more defective in catalysis than in protein folding. Moreover, the latter mutants, unlike the wild-type protein and the F103L variant, share a peculiar binding mode of dopa methyl ester consisting of formation of a quinonoid intermediate. This finding strongly suggests that their catalytic defects are mainly due to a misplacement of the substrate at the active site. Taken together, our results highlight the importance of the Arg347-Leu333-Asp345 hydrogen-bonds network in the catalysis of AADC and reveal the molecular basis for the pathogenicity of the variants R347. Following the above results, a therapeutic treatment for patients bearing the mutation R347G is proposed.
authors.affiliation: Department of Neurosciences, Biomedicine and Movement, University of Verona, Verona, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Montioli
authors.affiliation: Department of Biology and Biotechnology "Charles Darwin", La Sapienza University of Roma, Roma, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Paiardini
authors.affiliation: Developmental Neurosciences, UCL-Institute of Child Health, London, UK; Department of Neurology, Great Ormond Street Hospital, London, UK.
authors.forename: Manju A
authors.initials: MA
authors.lastname: Kurian
authors.affiliation: Department of Neurosciences, Biomedicine and Movement, University of Verona, Verona, Italy.
authors.forename: Mirco
authors.initials: M
authors.lastname: Dindo
authors.affiliation: Department of Neurosciences, Biomedicine and Movement, University of Verona, Verona, Italy.
authors.forename: Giada
authors.initials: G
authors.lastname: Rossignoli
authors.affiliation: Clinical Chemistry, Great Ormond Street Hospital, London, UK; Neurometabolic Unit, National Hospital of Neurology and Neurosurgery, UK.
authors.forename: Simon J R
authors.initials: SJR
authors.lastname: Heales
authors.affiliation: Neurometabolic Unit, National Hospital of Neurology and Neurosurgery, UK.
authors.forename: Simon
authors.initials: S
authors.lastname: Pope
authors.affiliation: Department of Neurosciences, Biomedicine and Movement, University of Verona, Verona, Italy.
authors.forename: Carla Borri
authors.initials: CB
authors.lastname: Voltattorni
authors.affiliation: Department of Neurosciences, Biomedicine and Movement, University of Verona, Verona, Italy. Electronic address: mita.bertoldi@univr.it.
authors.forename: Mariarita
authors.initials: M
authors.lastname: Bertoldi
mesh_terms: D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding
chemical_list: D001142:Aromatic-L-Amino-Acid Decarboxylases
keywords: AADC deficiency; Aromatic amino acid decarboxylase; Pathogenic variant; Pyridoxal 5′-phosphate