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: Fredj ben Bdira, Esperanza González, Paula Pluta, Ana Laín, Arantza Sanz-Parra, Juan Manuel Falcon-Perez, Oscar Millet
• Journal: Human molecular genetics (Hum Mol Genet) Vol. 23 Issue 21 Pg. 5805-13 (Nov 01 2014) ISSN: 1460-2083 [Electronic] England
• PMID: 24925316 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected].

Chemical References

• Uroporphyrinogen III Synthetase

Topics

• Amino Acid Substitution
• Catalysis
• Enzyme Activation
• Enzyme Stability
• Homeostasis
• Humans
• Intracellular Space (metabolism)
• Kinetics
• Models, Molecular
• Mutation
• Porphyria, Erythropoietic (enzymology, genetics, metabolism)
• Protein Conformation
• Protein Engineering
• Uroporphyrinogen III Synthetase (chemistry, genetics, metabolism)