Identifying a recombinant alkyldihydroxyacetonephosphate synthase suited for crystallographic studies |
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Authors: | Razeto Adelia Mattiroli Francesca Bossi Roberto Coda Alessandro Mattevi Andrea |
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Institution: | Dipartimento di Genetica e Microbiologia, Università di Pavia, via Ferrata 1, 27100 Pavia, Italy. |
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Abstract: | Alkyldihydroxyacetonephosphate is the building block for the biosynthesis of ether phospholipids, which are essential components of eukaryotic cell membranes and are involved in a variety of signaling processes. The metabolite is synthesized by alkyldihydroxyacetonephosphate synthase (ADPS), a peroxisomal flavoenzyme. Deficiency in ADPS activity causes rhizomelic chondrodysplasia punctata type 3, a very severe genetic disease. ADPS is unusual in that it uses a typical redox cofactor such as FAD to catalyze a non-redox reaction. With the goal of undertaking a structural investigation of the enzyme, we have characterized recombinant ADPS from different sources: Cavia porcellus, Drosophila melanogaster, Homo sapiens, Archaeoglobus fulgidus, and Dictyostelium discoideum. The protein from D. discoideum was found to be the best candidate for structural studies. We describe a protocol for expression and purification of large amounts of pure and stable enzyme in its holo (FAD-bound) form. A search of deletion mutants identified a protein variant that forms crystals diffracting up to 2A resolution. |
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Keywords: | Biosynthesis of ether phospholipids Flavin Peroxisomal disorder |
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