Now, Jeremy M. Baskin and co-workers from Cornell University report the directed evolution of an enzyme to enable the efficient conversion of phosphatidylcholine (PC) — the most abundant cellular membrane lipid — to a range of natural and unnatural lipids with diverse structures and functionalities. The work is based on a microbial phospholipase D (PLD) that can catalyse the exchange of phospholipid head groups by transphosphatidylation of PC with exogenous alcohols — to yield phosphatidyl alcohol (PAlc) lipids — or by hydrolysis with water — to yield the natural signalling lipid phosphatidic acid (PA) (pictured; BODIPY, boron dipyrromethene) — however, with limited activity and substrate scope.
Now, the researchers created a PLD mutant library and used their previously developed fluorescence-based assay termed Imaging PLD Activity with Clickable Alcohols via Transphosphatidylation (IMPACT) to screen for more active mutants in mammalian cells. After several rounds of screening and selection, variants with up to ~100× higher activity than the wild-type enzyme were identified. Importantly, the mutants could efficiently catalyse transphosphatidylation with several different primary and secondary alcohol substrates to form diverse PAlc lipids. It was found that higher protein stability was a significant factor for the improved efficiencies of the evolved enzymes and mutational studies indicated that the enhanced catalytic activity benefitted from cooperative effects of multiple amino acid substitutions. Moreover, the crystal structures of two evolved variants were solved, revealing an expanded active site. Finally, the researchers demonstrated that the PLD variants can be used for spatiotemporal editing of phospholipids in mammalian cells based on a previously developed optogenetics system. However, in that case, only the hydrolysis function of the enzyme was exploited.
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