PHOSPHOLIPASE-D FROM STREPTOMYCES-ANTIBIOTICUS - CLONING, SEQUENCING,EXPRESSION, AND RELATIONSHIP TO OTHER PHOSPHOLIPASES

The extracellular phospholipase D (PLD) gene from Streptomyces antibio ticus was cloned, sequenced, and expressed in Escherichia coli. Analys is of DNA sequence data revealed a putative ribosome-binding site and an open reading frame encoding a 556-amino-acid protein that included amino acid sequences obtained from the purified enzyme. The protein wa s expressed in an insoluble form in E. coli, but reacted with antibody against PLD. After solubilization of the protein with guanidine-HCl a nd 2-mercaptoethanol, subsequent dialysis restored the PLD activity. C omparison of the nucleotide sequence data with the N-terminal protein sequence indicates that this secreted protein is synthesized as a larg er precursor with a 47-amino-acid N-terminal extension to the mature e nzyme of 509 amino acids. The amino acid sequence of the S. antibiotic us PLD was extensively compared with other PLDs and phospholipase C (P LC). The deduced amino acid sequence of the cloned PLD was highly homo logous to PLDs from S. acinomyceticus and Streptomyces sp., and contai ned a conserved region with S. chromofuscus PLD. From comparisons of t he structural similarity and properties of the various PLDs, a classif ication of PLDs into two subgroups has been proposed and the highly co nserved region designated tentatively region X(PLD), which may be impo rtant in the catalytic function, has been identified. The homology com parison between our PLD and phosphatidylinositol-specific phospholipas e C (PI-PLC) is also discussed.