Characterization of Highly Functional Phospholipase D Produced by Streptoverticillium cinnamoneum

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Phospholipase D (PLD1), secreted into the culture medium of Streptoverticillium cinnnamoneum, has been purified to homogeneity and characterized. The Stv.PLD efficiently catalyzes both hydrolysis and transphosphatidylation of various phospholipids, including phosphatidylethanolamine (PE), phosphatidylcholinw (PC), and phosphatidylserine (PS). However, the substrate specificity differs between the two reactions ; PE serves as the most preferred substrate for the hydrolysis, but PC and PS are better substrates than PE for the transphosphatidylation. In addition, the transphosphatidylation but not the hydrolysis of PE and PC is markedly activated on the addition of metal ions, especially ALィイD13ィエD1ィイD2+ィエD2. Nucleotide and amino acid sequence determination of the Stv.PLD revealed the presence of common structural motifs identified in all PLD sequences from various species.The PLD located in the membrane fraction of Stv.(PLD2) is about 35-40-kDa-monomer enzyme, which is different from the secreted into the culture medium and the smallest molecule among the known PLDs. Anti-PLD1 polyclonal antibody did not cross react with PLD2 by Western blotting, suggesting that the overall structure of PLD2 is not similar to that of PLD1. PLD2 preferentially hydrolyzes PE over PC as a substrate. In the presence of ethanol as a donor of polar headgroup, PLD2 could not catalyze the transphosphatidylation reaction and exclusively produced phosphatidic acid. Thus, The enzymatic properties of PLD2 appear to be substantially different from those of PLD1.