Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C.
While searching for a phospholipase C (PLC) specific for phosphatidylcholine 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 secreted phosphatidylcholine-hydrolyzing PLC was purified by a combination of chromatographic procedures. As substrates, the enzyme preferred dipalmitoyl-phosphatidylcholine and 1-palmitoyl-2-arachidonoyl-phosphatidylcholine over phosphatidylinositol. The active enzyme is a monomer of approximately 40 kDa. As for other bacterial PLCs, the enzyme requires Ca2+ and Zn2+ for activity; dithiothreitol affected the activity due to its chelation of Zn2+, but this inhibition could be compensated for by addition of ZnCl2. The compound D609, described to selectively inhibit phosphatidylcholine-specific PLCs, caused half-inhibition of the P. fluorescens enzyme at approximately 420 microM, while 50-fold lower concentrations similarly affected PLCs from Bacillus cereus and Clostridium perfringens. Partial peptide sequences obtained 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 encoding the PLC. There is no significant sequence homology to other PLCs, suggesting that the P. fluorescens enzyme represents a distinct subclass of bacterial 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 coding sequence.
Preuss
I
I
Ruprecht-Karls-Universität Heidelberg, Biochemie-Zentrum Heidelberg, Biologische Chemie, Heidelberg, Germany.
Kaiser
I
I
Gehring
U
U
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010713:Phosphatidylcholines / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011551:Pseudomonas fluorescens / Q000201:enzymology; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004269:DNA, Bacterial; D010713:Phosphatidylcholines; D010738:Type C Phospholipases