title: A novel class of microbial phosphocholine-specific phospholipases C.
abstract: In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.
authors.affiliation: Department of Microbiology, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Box B-175, Denver, CO 80262, USA.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Stonehouse
authors.forename: Adela
authors.initials: A
authors.lastname: Cota-Gomez
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Parker
authors.forename: Wesley E
authors.initials: WE
authors.lastname: Martin
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Hankin
authors.forename: Robert C
authors.initials: RC
authors.lastname: Murphy
authors.forename: Weibin
authors.initials: W
authors.lastname: Chen
authors.forename: Kheng B
authors.initials: KB
authors.lastname: Lim
authors.forename: Murray
authors.initials: M
authors.lastname: Hackett
authors.forename: Adriana I
authors.initials: AI
authors.lastname: Vasil
authors.forename: Michael L
authors.initials: ML
authors.lastname: Vasil
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002794:Choline; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D006461:Hemolysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D010743:Phospholipids; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C; D002794:Choline; D002118:Calcium