Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane protein

•Human DHAPAT is associated to peroxisomal membrane through the N-terminal region.•Recombinant human DHAPAT expressed and purified from P. pastoris cells is active.•Evidence of the in vitro reconstitution of DHAPAT/ADPS enzymatic complex.

Although the precise functions of ether phospholipids are still poorly understood, significant alterations in their physiological levels are associated either to inherited disorders or to aggressive metastatic cancer. The essential precursor, alkyl-dihydroxyacetone phosphate (DHAP), for all ether phospholipids species is synthetized in two consecutive reactions performed by two enzymes sitting on the inner side of the peroxisomal membrane. Here, we report the characterization of the recombinant human DHAP acyl-transferase, which performs the first step in alkyl-DHAP synthesis. By exploring several expression systems and designing a number of constructs, we were able to purify the enzyme in its active form and we found that it is tightly bound to the membrane through the N-terminal residues.