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Biochemistry. 2015 Sep 15;54(36):5617-31. doi: 10.1021/acs.biochem.5b00407. Epub 2015 Sep 2.

Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release.

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Department of Molecular Medicine, Morsani College of Medicine, University of South Florida , Tampa, Florida 33612, United States.
Assistance Publique-Hôpitaux de Paris, Centre Français des Porphyries, Hôpital Louis Mourier , 178 rue des Renouillers, 92701 Colombes Cedex, France.
INSERM U1149, CNRS ERL 8252, Centre de Recherche sur l'inflammation, 16 rue Henri Huchard, 75018, Université Paris Diderot, Site Bichat, 75018 Paris, France.
Laboratory of Excellence, GR-Ex , Paris, France.
Department of Chemistry, University of South Florida , Tampa, Florida 33612, United States.


Regulation of 5-aminolevulinate synthase (ALAS) is at the origin of balanced heme production in mammals. Mutations in the C-terminal region of human erythroid-specific ALAS (hALAS2) are associated with X-linked protoporphyria (XLPP), a disease characterized by extreme photosensitivity, with elevated blood concentrations of free protoporphyrin IX and zinc protoporphyrin. To investigate the molecular basis for this disease, recombinant hALAS2 and variants of the enzyme harboring the gain-of-function XLPP mutations were constructed, purified, and analyzed kinetically, spectroscopically, and thermodynamically. Enhanced activities of the XLPP variants resulted from increases in the rate at which the product 5-aminolevulinate (ALA) was released from the enzyme. Circular dichroism spectroscopy revealed that the XLPP mutations altered the microenvironment of the pyridoxal 5'-phosphate cofactor, which underwent further and specific alterations upon succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon binding of ALA to the XLPP variants demonstrated that the protein conformational transition step associated with product release was predominantly affected. Of relevance is the fact that XLPP could also be modeled in cell culture. We propose that (1) the XLPP mutations destabilize the succinyl-CoA-induced hALAS2 closed conformation and thus accelerate ALA release, (2) the extended C-terminus of wild-type mammalian ALAS2 provides a regulatory role that allows for allosteric modulation of activity, thereby controlling the rate of erythroid heme biosynthesis, and (3) this control is disrupted in XLPP, resulting in porphyrin accumulation.

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