Identifying a recombinant alkyldihydroxyacetonephosphate synthase suited for crystallographic studies.
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.
Razeto
Adelia
A
Dipartimento di Genetica e Microbiologia, Università di Pavia, via Ferrata 1, 27100 Pavia, Italy.
Mattiroli
Francesca
F
Bossi
Roberto
R
Coda
Alessandro
A
Mattevi
Andrea
A
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
D017931:DNA Primers; D011994:Recombinant Proteins; D019883:Alkyl and Aryl Transferases; C019654:alkylglycerone-phosphate synthase