A novel class of microbial phosphocholine-specific phospholipases C

Area of Science:

• Biochemistry
• Microbiology
• Enzymology

Background:

• Pseudomonas aeruginosa produces a hemolytic phospholipase C (PLC).
• This PLC belongs to a novel superfamily of PLC/phosphatase enzymes.
• Related proteins are found in Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis, and Burkholderia pseudomallei.

Purpose of the Study:

• To purify and characterize the hemolytic phospholipase C (PLC) from Pseudomonas aeruginosa.
• To investigate the enzymatic and hemolytic properties of the purified enzyme complex.
• To elucidate the substrate specificity and inhibition profile of the enzyme.

Main Methods:

• Overexpression of the plcHR1,2 operon.
• Ion exchange chromatography and native preparative polyacrylamide gel electrophoresis for purification.
• MALDI-TOF and LCMS for protein identification and stoichiometry.
• Western blot analysis for complex confirmation.
• Enzymatic and hemolytic activity assays.

Main Results:

• A 1,500-fold purification of the hemolytic PLC from P. aeruginosa was achieved.
• The purified enzyme exists as a heterodimeric complex, PlcHR2, composed of PlcH and PlcR2.
• PlcHR2 is active on choline-containing phospholipids, including phosphatidylcholine (PC) and sphingomyelin (SM).
• Calcium binds to PlcHR2, inhibiting hemolytic activity without affecting enzymatic activity.
• The chaperone PlcR2 influences both enzymatic and hemolytic properties of PlcH.

Conclusions:

• The purified PlcHR2 complex represents a novel enzyme with specific activity towards choline-containing phospholipids.
• Calcium's differential effect on enzymatic versus hemolytic activity suggests distinct functional roles.
• Members of this PC-PLC and phosphatase family utilize a novel mechanism for substrate recognition and hydrolysis.