Abstract
Plasmalogens are an abundant class of glycerophospholipids in the mammalian body, with special occurrence in the brain and in immune cell membranes. Plasmanylethanolamine desaturase (PEDS1) is the final enzyme of plasmalogen biosynthesis, which introduces the characteristic 1-O-alk-1'-enyl double bond. The recent sequence identification of PEDS1 as transmembrane protein 189 showed that its protein sequence is related to a special class of plant desaturases (FAD4), with whom it shares a motif of 8 conserved histidines, which are essential for the enzymatic activity. In the present work, we wanted to gain more insight into the sequence-function relationship of this enzyme and mutated to alanine additional 28 amino acid residues of murine plasmanylethanolamine desaturase including those 20 residues, which are also totally conserved-in addition to the eight-histidine-motif-among the animal PEDS1 and plant FAD4 plant desaturases. We measured the enzymatic activity by transient transfection of tagged murine PEDS1 expression clones to a PEDS1-deficient human HAP1 cell line by monitoring of labeled plasmalogens formed from supplemented 1-O-pyrenedecyl-sn-glycerol in relation to recombinant protein expression. Surprisingly, only a single mutation, namely aspartate 100, led to a total loss of PEDS1 activity. The second strongest impact on enzymatic activity had mutation of phenylalanine 118, leaving only 6% residual activity. A structural model obtained by homology modelling to available structures of stearoyl-CoA reductase predicted that this aspartate 100 residue interacts with histidine 96, and phenylalanine 118 interacts with histidine 187, both being essential histidines assumed to be involved in the coordination of the di-metal center of the enzyme.