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

Atsushi Masayama, Tetsuya Takahashi, Kaori Tsukada, Seigo Nishikawa, Rie Takahashi, Masaatsu Adachi, Kazushi Koga, Atsuo Suzuki, Takashi Yamane, Hideo Nakano, Yugo Iwasaki

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

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 Pl-synthesizing mutant PLDs.One of these mutant 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 Pls 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 Pl synthesis by Streptomyces PLD.

Original languageEnglish
Pages (from-to)974-981
Number of pages8
JournalChemBioChem
Volume9
Issue number6
DOIs
Publication statusPublished - 2008 Apr 14
Externally publishedYes

Keywords

  • Enzymes
  • High-throughput screening
  • Molecular evolution
  • Phosphatidylinositol
  • Phospholipases

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry

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  • Cite this

    Masayama, A., Takahashi, T., Tsukada, K., Nishikawa, S., Takahashi, R., Adachi, M., Koga, K., Suzuki, A., Yamane, T., Nakano, H., & Iwasaki, Y. (2008). Streptomyces phospholipase D mutants with altered substrate specificity capable of phosphatidylinositol synthesis. ChemBioChem, 9(6), 974-981. https://doi.org/10.1002/cbic.200700528