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Published on: January 10, 2018
Identifying a recombinant alkyldihydroxyacetonephosphate synthase suited for crystallographic studies
Adelia Razeto1, Francesca Mattiroli, Roberto Bossi
1Dipartimento di Genetica e Microbiologia, Università di Pavia, via Ferrata 1, 27100 Pavia, Italy.
Protein Expression and Purification
|July 3, 2007
Summary
Alkyldihydroxyacetonephosphate synthase (ADPS) is crucial for ether phospholipid synthesis. Researchers purified ADPS from various species, identifying a Dictyostelium discoideum variant suitable for structural studies and crystal formation.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Ether phospholipids are vital for eukaryotic cell membranes and signaling.
- Alkyldihydroxyacetonephosphate synthase (ADPS) synthesizes a key precursor, alkyldihydroxyacetonephosphate.
- ADPS deficiency leads to severe genetic disorders like rhizomelic chondrodysplasia punctata type 3.
Purpose of the Study:
- To characterize recombinant ADPS from diverse species for structural investigation.
- To identify the optimal ADPS source and develop a purification protocol for structural studies.
- To obtain a high-resolution structure of ADPS to understand its unique catalytic mechanism.
Main Methods:
- Recombinant expression and purification of ADPS from Cavia porcellus, Drosophila melanogaster, Homo sapiens, Archaeoglobus fulgidus, and Dictyostelium discoideum.
- Characterization of enzyme stability and cofactor (FAD) binding.
- Screening of deletion mutants to identify variants suitable for crystallization.
Main Results:
- ADPS from Dictyostelium discoideum was selected as the best candidate for structural studies.
- An efficient protocol for large-scale expression and purification of holo-ADPS was established.
- A deletion mutant was identified that forms crystals diffracting to 2A resolution.
Conclusions:
- The study provides a robust method for obtaining pure, stable ADPS for structural analysis.
- The identified crystal-forming mutant is promising for elucidating the structure of ADPS.
- Structural insights into ADPS could advance understanding of ether phospholipid biosynthesis and related diseases.
