A novel class of microbial phosphocholine-specific phospholipases C

被引:78
作者
Stonehouse, MJ
Cota-Gomez, A
Parker, SK
Martin, WE
Hankin, JA
Murphy, RC
Chen, WB
Lim, KB
Hackett, M
Vasil, AI
Vasil, ML
机构
[1] Univ Colorado, Hlth Sci Ctr, Dept Microbiol, Denver, CO 80262 USA
[2] Natl Jewish Med & Res Ctr, Div Basic Sci, Dept Pediat, Denver, CO 80206 USA
[3] Univ Washington, Dept Med Chem, Seattle, WA 98195 USA
关键词
D O I
10.1046/j.1365-2958.2002.03194.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In this report we describe the 1500-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 plcHR(1,2) 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 (PlcR(2)) and 78 417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR(2) are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR(2). PlcHR(2) 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 PlcHR(2) nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR(2), and the PlcHR(2) complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR(2). In addition to being required for the secretion of PlcH, the chaperone PlcR(2) 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.
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页码:661 / 676
页数:16
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