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

Chembiochem : a European Journal of Chemical Biology
Atsushi MasayamaYugo Iwasaki

Abstract

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...Continue Reading

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Mar 14, 2008·Chembiochem : a European Journal of Chemical Biology·Atsushi MasayamaYugo Iwasaki

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Citations

Jun 22, 2012·Protein Engineering, Design & Selection : PEDS·Jasmina DamnjanovićYugo Iwasaki
May 4, 2013·Journal of Bioscience and Bioengineering·Jasmina Damnjanović, Yugo Iwasaki
Mar 17, 2010·Journal of Bioscience and Bioengineering·Akari OzakiYugo Iwasaki
Mar 25, 2016·Journal of Bioscience and Bioengineering·Michiko MurakiYugo Iwasaki
Jun 16, 2016·Progress in Lipid Research·Katja ZornUwe T Bornscheuer
Apr 20, 2017·Journal of Lipid Research·Satu HänninenPentti Somerharju
Apr 17, 2019·Bioprocess and Biosystems Engineering·Hai-Juan HouJin-Song Shi
Aug 10, 2019·Protein Engineering, Design & Selection : PEDS·Jasmina DamnjanovićYugo Iwasaki
May 15, 2021·Critical Reviews in Biotechnology·Peng ZhangJin-Song Shi

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