title: Tuning intracellular homeostasis of human uroporphyrinogen III synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria.
abstract: Congenital erythropoietic porphyria (CEP) results from a deficiency in uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.
authors.affiliation: Structural Biology Unit and Metabolomics Unit, CIC bioGUNE, Bizkaia Technology Park, Building 800-801A, Derio 48160, Spain and.
authors.forename: Fredj
authors.initials: F
authors.lastname: ben Bdira
authors.affiliation: Structural Biology Unit and Metabolomics Unit, CIC bioGUNE, Bizkaia Technology Park, Building 800-801A, Derio 48160, Spain and.
authors.forename: Esperanza
authors.initials: E
authors.lastname: González
authors.affiliation: Structural Biology Unit and Metabolomics Unit, CIC bioGUNE, Bizkaia Technology Park, Building 800-801A, Derio 48160, Spain and.
authors.forename: Paula
authors.initials: P
authors.lastname: Pluta
authors.affiliation: Structural Biology Unit and Metabolomics Unit, CIC bioGUNE, Bizkaia Technology Park, Building 800-801A, Derio 48160, Spain and.
authors.forename: Ana
authors.initials: A
authors.lastname: Laín
authors.affiliation: Structural Biology Unit and Metabolomics Unit, CIC bioGUNE, Bizkaia Technology Park, Building 800-801A, Derio 48160, Spain and.
authors.forename: Arantza
authors.initials: A
authors.lastname: Sanz-Parra
authors.affiliation: Structural Biology Unit and Metabolomics Unit, CIC bioGUNE, Bizkaia Technology Park, Building 800-801A, Derio 48160, Spain and IKERBASQUE, Basque Foundation for Science, Bilbao 48011, Spain.
authors.forename: Juan Manuel
authors.initials: JM
authors.lastname: Falcon-Perez
authors.affiliation: Structural Biology Unit and Metabolomics Unit, CIC bioGUNE, Bizkaia Technology Park, Building 800-801A, Derio 48160, Spain and omillet@cicbiogune.es.
authors.forename: Oscar
authors.initials: O
authors.lastname: Millet
mesh_terms: D019943:Amino Acid Substitution; D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D006706:Homeostasis; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D017092:Porphyria, Erythropoietic / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D014576:Uroporphyrinogen III Synthetase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D014576:Uroporphyrinogen III Synthetase