title: Identifying a recombinant alkyldihydroxyacetonephosphate synthase suited for crystallographic studies.
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.
authors.affiliation: Dipartimento di Genetica e Microbiologia, Università di Pavia, via Ferrata 1, 27100 Pavia, Italy.
authors.forename: Adelia
authors.initials: A
authors.lastname: Razeto
authors.forename: Francesca
authors.initials: F
authors.lastname: Mattiroli
authors.forename: Roberto
authors.initials: R
authors.lastname: Bossi
authors.forename: Alessandro
authors.initials: A
authors.lastname: Coda
authors.forename: Andrea
authors.initials: A
authors.lastname: Mattevi
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C019654:alkylglycerone-phosphate synthase