N-terminal domain swapping contributes to phosphatidylcholine:diacylglycerol cholinephosphotransferase function.

Plant oils are widely used in the food, fuel, and oleochemical industries, with their chemical properties and applications largely determined by fatty acid composition. Phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), a plant-exclusive, multi-spanning transmembrane enzyme, catalyses the interconversion between diacylglycerol and phosphatidylcholine and plays a key role in shaping oil fatty acid profiles in plants. Despite its importance, the structure-function relationship of PDCT remains poorly understood. Our recent structural modelling suggests that PDCT may function as a dimer through swapping of the N-terminal region between protomers. In this study, we used soybean (Glycine max) PDCT1 to further examine the terminal regions and key residues in the predicted swapped N-terminal transmembrane region required for PDCT activity. Our results reveal that the disordered region at the N-terminus, and the less-conserved C-terminus, are both dispensable for catalysis, while conserved residues Glu75 and Asp89 within the swapped N-terminal transmembrane region are essential for enzyme activity and/or stability. Notably, co-expression of the inactive E75A N-terminal mutant and the inactive D230A C-terminal catalytic site mutant partially restored PDCT activity, providing support for the proposed domain-swapping mechanism. Together, this study provides new insights into the catalytic mechanisms of PDCT and may inform future efforts to exploit PDCT in plant oil engineering.