Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C

While searching for a phospholipase C (PLC) specific for phosphatidylcholin e in mammalian tissues, we came across such an activity originating from a contamination of Pseudomonas fluorescens. This psychrophilic bacterium was found to contaminate placental extracts upon processing in the cold. The se creted phosphatidylcholine-hydrolyzing PLC was purified by a combination of chromatographic procedures. As substrates, the enzyme preferred dipalmitoy l-phosphatidylcholine and 1-palmitoyl-2-arachidonoyl-phosphatidylcholine ov er phosphatidylinositol. The active enzyme is a monomer of approximate to 4 0 kDa. As for other bacterial PLCs, the enzyme requires Ca2+ and Zn2+ for a ctivity; dithiothreitol affected the activity due to its chelation of Zn2+, but this inhibition could be compensated for by addition of ZnCl2. The com pound D609, described to selectively inhibit phosphatidylcholine-specific P LCs, caused half-inhibition of the P. fluorescens enzyme at approximate to 420 muM, while 50-fold lower concentrations similarly affected PLCs from Ba cillus cereus and Clostridium perfringens. Partial peptide sequences obtain ed from the pure P. fluorescens enzyme after tryptic cleavage were used to clone a DNA fragment of 3.5 kb from a P. fluorescens gene library prepared from our laboratory isolate. It contains an ORF of 1155 nucleotides encodin g the PLC. There is no significant sequence homology to other PLCs, suggest ing that the P. fluorescens enzyme represents a distinct subclass of bacter ial PLCs. The protein lacks cysteine residues and consequently contains no disulfide bonds. Interestingly, P. fluorescens reference strain DSMZ 50090 is devoid of the PLC activity described here as well as of the relevant cod ing sequence.