Isolation of phospholipase D mutants having phosphatidylinositol-synthesizing activity with positional specificity on myo-inositol.

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.

Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis.

The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10,000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLD was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD.