Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane protein.
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.
Department of Biology and Biotechnology, University of Pavia, V. Ferrata 9, 27100, Pavia, Italy.
Valentina
V
Piano
Department of Biology and Biotechnology, University of Pavia, V. Ferrata 9, 27100, Pavia, Italy.
Simone
S
Nenci
Department of Biology and Biotechnology, University of Pavia, V. Ferrata 9, 27100, Pavia, Italy.
Francesca
F
Magnani
Department of Biosciences, University of Milan, V. Celoria 26, 20133, Milan, Italy.
Alessandro
A
Aliverti
Department of Biology and Biotechnology, University of Pavia, V. Ferrata 9, 27100, Pavia, Italy. Electronic address: andrea.mattevi@unipv.it.
Andrea
A
Mattevi
D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D020675:Peroxisomes / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D008565:Membrane Proteins; D011994:Recombinant Proteins; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase
Ether-phospholipids; Membrane protein; Peroxisomal disorder; Rhizomelic chondrodysplasia punctata