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Genomic structure, cloning and expression of two phospholipase D isoenzymes from white cabbage
Author(s) -
Schäffner Ines,
Rücknagel KarlPeter,
Mansfeld Johanna,
UlbrichHofmann Renate
Publication year - 2002
Publication title -
european journal of lipid science and technology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.614
H-Index - 94
eISSN - 1438-9312
pISSN - 1438-7697
DOI - 10.1002/1438-9312(200202)104:2<79::aid-ejlt79>3.0.co;2-c
Subject(s) - pld2 , phospholipase d , biochemistry , enzyme , complementary dna , cloning (programming) , biology , phospholipase , microbiology and biotechnology , isozyme , chemistry , gene , phospholipid , phosphatidic acid , computer science , programming language , membrane
Phospholipase D (PLD) from cabbage is interesting as biocatalyst in phospholipid transformation. To provide the basis for genetic engineering of the enzyme, gene cloning and sequencing were carried out. We have recently identified two isoenzymes, PLD1 and PLD2, on the basis of their cDNAs, here we describe their genomic structure consisting of 3404 and 3614 bp, respectively. Based on their sequence, PLD1 and PLD2 can be assigned to the α‐type of plant PLDs, they contain two HKD motifs and the C2 domain with a phosphatidylinositol 4,5‐bisphosphate (PIP 2 ) binding motif. Starting from pld1 cDNA, expression studies were carried out. Whereas expression using constructs with StrepTactin or Glutathion S‐transferase tags was not successful, soluble active enzyme was produced from constructs without tag. pld2 was expressed accordingly. Both enzymes were purified by Ca 2+ ‐mediated hydrophobic interaction chromatography to high purity. N‐terminal sequencing of PLD1 and PLD2 revealed that the Met‐free N‐termini of both enzymes correspond to sequences derived from the coding region of the pld 1 and pld 2 genes, respectively. Both recombinant enzymes showed highest hydrolytic activities at pH 5.5 to 5.6, independent of Ca 2+ concentration (10—100 mM). The optimum Ca 2+ concentration was 45 mM for PLD1 and PLD2. Both enzymes showed comparable activities in hydrolysis and transphosphatidylation of phospholipids.