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Isolation of Phospholipase D Mutants Having Phosphatidylinositol‐Synthesizing Activity with Positional Specificity on myo ‐Inositol
Author(s) -
Masayama Atsushi,
Tsukada Kaori,
Ikeda Chika,
Nakano Hideo,
Iwasaki Yugo
Publication year - 2009
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200800651
Subject(s) - mutant , phosphatidylinositol , inositol , phospholipase c , stereochemistry , phospholipase d , pi , biochemistry , phospholipase , phosphatidylcholine , enzyme , chemistry , biology , phospholipid , signal transduction , gene , receptor , membrane
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.