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Keywords:

  • enzymes;
  • high-throughput screening;
  • phosphatidylinositol;
  • phospholipases;
  • regioselectivity

Abstract

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Enzyme-mediated synthesis of phosphatidylinositol: Engineered phospholipase D enzymes enable the synthesis of phosphatidylinositol by transphosphatidylation. The 1- or 3-hydroxy group of myo-inositol is selectively reacted.

Phospholipase D (PLD) mutants that have phosphatidylinositol (PI)-synthesizing activity with positional selectivity towards 1- or 3-OH groups of myo-inositol have been isolated. A mutant PLD library, in which site-directed saturation mutations were introduced in vitro at positions 187, 191, and 385 of the wild-type PLD of Streptomyces antibioticus, was screened for PI-synthesizing mutants. TLC and HPLC analyses of the PI synthesized by the isolated mutant PLDs revealed that three mutants, namely 187D/191Y/385R (DYR), 187A/191Y/385R (AYR), and 187M/191Y/385R (MYR), selectively generated 1- or 3-PI among the other possible PI positional isomers. Taking into account the consensus sequence of the three mutants, a series of mutants, 187X/191Y/385R (XYR), was constructed and analyzed. Almost all the XYR mutants generated 1(3)-PI selectively, thus suggesting that the Y385R mutation contributed to the selectivity for the 1(3)-PI synthesis. The XYR mutants showed similar phosphatidylcholine-hydrolyzing activity among the mutants, but the PI-synthesizing activities were different depending on the amino acid at position 187. In particular, aromatic amino acids at position 187 greatly reduced the PI-synthesizing activity. The ratios of 1-PI versus 3-PI in the PIs synthesized with the XYR mutants were analyzed by selective hydrolysis with PI-specific phospholipase C. It was found that 187H/191Y/385R (HYR) generated 1-PI more than 3-PI (ratio=7:3), whereas 187T/191Y/385R (TYR) generated 1-PI less than 3-PI (ratio=2:8). This confirmed that the amino acid at position 187 determined the selectivity between 1-PI and 3-PI formation.