Bacterial phospholipase A: structure and function of an integral membrane phospholipase

Snijder, H.J.; Dijkstra, Bauke W.

doi:10.1016/s1388-1981(00)00113-x

Cited by 83 publications

(70 citation statements)

References 48 publications

(61 reference statements)

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“…Recently, noble gas soaking has also been exploited to successfully map the detergent in porin crystals by lowresolution X-ray diffraction (Sauer et al, 2002). Here, we have employed single crystal neutron diffraction to elucidate the organisation of the b-octylglucoside (b-OG) Outer membrane phospholipase A is an integral membrane phospholipase, which is present in many Gram-negative bacteria (reviewed in Snijder and Dijkstra, 2000). Its 3D-structure consists of a 12-stranded antiparallel b-barrel with a convex and a flat side.…”

Section: Introductionmentioning

confidence: 99%

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Snijder¹,

Timmins²,

Kalk³

et al. 2003

Journal of Structural Biology

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The structure of the detergent in crystals of outer membrane phospholipase A (OMPLA) has been determined using neutron diffraction contrast variation. Large crystals were soaked in stabilising solutions, each containing a different H 2 O=D 2 O contrast. From the neutron diffraction at five contrasts, the 12 A A resolution structure of the detergent micelle around the protein molecule was determined. The hydrophobic b-barrel surfaces of the protein molecules are covered by rings of detergent. These detergent belts are fused to neighbouring detergent rings forming a continuous three-dimensional network throughout the crystal. The thickness of the detergent layer around the protein varies from 7-20 A A. The enzymeÕs active site is positioned just outside the hydrophobic detergent zone and is thus in a proper location to catalyse the hydrolysis of phospholipids in a natural membrane. Although the dimerisation face of OMPLA is covered with detergent, the detergent density is weak near the exposed polar patch, suggesting that burying this patch in the enzymeÕs dimer interface may be energetically favourable. Furthermore, these results indicate a crucial role for detergent coalescence during crystal formation and contribute to the understanding of membrane protein crystallisation.

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Section: Introductionmentioning

confidence: 99%

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Snijder¹,

Timmins²,

Kalk³

et al. 2003

Journal of Structural Biology

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“…Activation of the enzyme requires calcium (60) and its dimerization (19,20) and may require the presence of phospholipids in the outer leaflet of the outer membrane (18,44,53). In normally growing cells, OM-PLA is in the monomeric form and the outer leaflet is believed to be composed entirely of lipopolysaccharides.…”

mentioning

confidence: 99%

Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degP Mutants and Induces the Cpx and ς ^E Extracytoplasmic Stress Responses

et al. 2001

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DegP is a periplasmic protease that is a member of both the E and Cpx extracytoplasmic stress regulons of Escherichia coli and is essential for viability at temperatures above 42°C. [U-14 C]acetate labeling experiments demonstrated that phospholipids were degraded in degP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, ␤-lactamase, and ␤-galactosidase assays as well as sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degP cells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42°C, displayed increased viability at 44°C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and E regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype of degP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and E regulons rather than by inactivating the phospholipase per se.Extracytoplasmic stress, such as that caused by heat shock or the overproduction of outer membrane proteins in Escherichia coli, is believed to be caused by the accumulation and aggregation of denatured and misfolded proteins in the membranes and periplasm. Under these conditions the Cpx two-component signal transduction pathway and the alternative sigma factor E direct the synthesis of several proteins that are involved in the degradation and refolding of these denatured and misfolded periplasmic proteins, leading to alleviation of the stress (for a review, see reference 50).The rpoE gene encoding E is essential for the viability of cells at all temperatures (22).E is known to direct the transcription of degP (htrA), fkpA, rpoE, rpoH, and many others (12,14,15,26,34). DegP is a protease/chaperone that digests abnormal proteins in the periplasm and has been demonstrated to be necessary for cell viability at temperatures of 42°C and above (33,35,55,57,58,59), and FkpA is a peptidyl prolyl cis/trans isomerase (28, 41). RseA, RseB, and RseC are involved in regulating the transcription of genes in the E regulon (21, 42). Under heat shock conditions or upon overexpression of outer membrane proteins, denatured and misfolded proteins in the periplasm are sensed by RseA and/or RseB (38). E is then released by the cytoplasmic domain, allowing it to direct transcription of the genes in the E regulon.

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“…Although macrophage or amoeba cell infection rates were not influenced by the presence or absence of PlaB, guinea pig infection experiments show its importance for Legionella replication and dissemination in vivo (1). 3 PlaB does not comprise hydrophobic stretches to form ␤-barrel structures; nevertheless, it could resemble E. coli or Helicobacter pylori outer membrane phospholipase A function, to allow bacterial adaptation through membrane alteration during the infection process (31,32). However, PlaB does not show protein homology to outer membrane phospholipase A enzymes or to any other phospholipase described so far.…”

Section: Discussionmentioning

confidence: 98%

Phospholipase PlaB of Legionella pneumophila Represents a Novel Lipase Family

Bender

Rydzewski

Broich

et al. 2009

Journal of Biological Chemistry

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Legionella pneumophila possesses several phospholipases capable of host cell manipulation and lung damage. Recently, we discovered that the major cell-associated hemolytic phospholipase A (PlaB) shares no homology to described phospholipases and is dispensable for intracellular replication in vitro. Nevertheless, here we show that PlaB is the major lipolytic activity in L. pneumophila cell infections and that PlaB utilizes a typical catalytic triad of Ser-Asp-His for effective hydrolysis of phospholipid substrates. Crucial residues were found to be located within the N-terminal half of the protein, and amino acids embedding these active sites were unique for PlaB and homologs. We further showed that catalytic activity toward phosphatidylcholine but not phosphatidylglycerol is directly linked to hemolytic potential of PlaB. Although the function of the prolonged PlaB C terminus remains to be elucidated, it is essential for lipolysis, since the removal of 15 amino acids already abolishes enzyme activity. Additionally, we determined that PlaB preferentially hydrolyzes long-chain fatty acid substrates containing 12 or more carbon atoms. Since phospholipases play an important role as bacterial virulence factors, we examined cell-associated enzymatic activities among L. pneumophila clinical isolates and non-pneumophila species. All tested clinical isolates showed comparable activities, whereas of the non-pneumophila species, only Legionella gormanii and Legionella spiritensis possessed lipolytic activities similar to those of L. pneumophila and comprised plaB-like genes. Interestingly, phosphatidylcholine-specific phospholipase A activity and hemolytic potential were more pronounced in L. pneumophila. Therefore, hydrolysis of the eukaryotic membrane constituent phosphatidylcholine triggered by PlaB could be an important virulence tool for Legionella pathogenicity.

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Bacterial phospholipase A: structure and function of an integral membrane phospholipase

Cited by 83 publications

References 48 publications

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degP Mutants and Induces the Cpx and ς ^E Extracytoplasmic Stress Responses

Phospholipase PlaB of Legionella pneumophila Represents a Novel Lipase Family

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Bacterial phospholipase A: structure and function of an integral membrane phospholipase

Cited by 83 publications

References 48 publications

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degP Mutants and Induces the Cpx and ς E Extracytoplasmic Stress Responses

Phospholipase PlaB of Legionella pneumophila Represents a Novel Lipase Family

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Absence of the Outer Membrane Phospholipase A Suppresses the Temperature-Sensitive Phenotype of Escherichia coli degP Mutants and Induces the Cpx and ς ^E Extracytoplasmic Stress Responses