Identifying a recombinant alkyldihydroxyacetonephosphate synthase suited for crystallographic studies

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• 作者：Adelia Razeto ; Francesca Mattiroli ; Roberto Bossi ; Aless ; ro Coda ; Andrea Mattevi
• 关键词：Biosynthesis of ether phospholipids ; Flavin ; Peroxisomal disorder
• 刊名：Protein Expression and Purification
• 出版年：2007
• 出版时间：October 2007
• 年：2007
• 卷：55
• 期：2
• 页码：343-351
• 全文大小：1949 K

文摘

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 2 Å resolution.