Genomic structure, cloning and expression of two phospholipase D isoenzymes from white cabbage

Abstract

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 (PIP2) 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 Ca2+-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 pld1 and pld2 genes, respectively. Both recombinant enzymes showed highest hydrolytic activities at pH 5.5 to 5.6, independent of Ca2+ concentration (10—100 mM). The optimum Ca2+ concentration was 45 mM for PLD1 and PLD2. Both enzymes showed comparable activities in hydrolysis and transphosphatidylation of phospholipids.