title: Uroporphyrinogen III synthase mutations related to congenital erythropoietic porphyria identify a key helix for protein stability.
abstract: In the present study we have investigated deleterious mutants in the uroporphyrinogen III synthase (UROIIIS) that are related to the congenital erythropoietic porphyria (CEP). The 25 missense mutants found in CEP patients have been cloned, expressed, and purified. Their enzymatic activities have been measured relative to wild-type UROIIIS activity. All mutants retain measurable activity, consistent with the recessive character of the disease. Most of the mutants with a significant decrease in activity involve residues likely associated in binding. However, other mutants are fully active, indicating that different mechanisms may contribute to enzyme missfunction. UROIIIS is a thermolabile enzyme undergoing irreversible denaturation. The unfolding kinetics of wild-type UROIIIS and the suite of mutants have been monitored by circular dichroism. This analysis allowed the identification of a helical region in the molecule, essential to retain the kinetic stability of the folded conformation. C73R is found in one-third of CEP patients, and Cys73 is part of this helix. The integrated analysis of the enzymatic activity and kinetic stability data is used to gain insight in the relationship between defects in UROIIIS sequence and CEP.
authors.affiliation: Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, Building 800, 48160 Derio, Spain.
authors.forename: Arola
authors.initials: A
authors.lastname: Fortian
authors.forename: David
authors.initials: D
authors.lastname: Castaño
authors.forename: Gabriel
authors.initials: G
authors.lastname: Ortega
authors.forename: Ana
authors.initials: A
authors.lastname: Laín
authors.forename: Miquel
authors.initials: M
authors.lastname: Pons
authors.forename: Oscar
authors.initials: O
authors.lastname: Millet
mesh_terms: D000483:Alleles; D002942:Circular Dichroism; D004795:Enzyme Stability; D005787:Gene Frequency; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D020125:Mutation, Missense; D017092:Porphyria, Erythropoietic / Q000201:enzymology / Q000235:genetics*; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014576:Uroporphyrinogen III Synthetase / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014576:Uroporphyrinogen III Synthetase