Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids

doi:10.1016/S1388-1981(99)00153-5

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids

Volume 1441, Issues 2–3, 23 November 1999, Pages 237-254

https://doi.org/10.1016/S1388-1981(99)00153-5 Get rights and content

Abstract

The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) is a small, water-soluble enzyme that cleaves the natural membrane lipids PI, lyso-PI, and glycosyl-PI. The crystal structure, NMR and enzymatic mechanism of bacterial PI-PLCs are reviewed. These enzymes consist of a single domain folded as a (βα)₈-barrel (TIM barrel), are calcium-independent, and interact weakly with membranes. Sequence similarity among PI-PLCs from different bacterial species is extensive, and includes the residues involved in catalysis. Bacterial PI-PLCs are structurally similar to the catalytic domain of mammalian PI-PLCs. Comparative studies of both prokaryotic and eukaryotic isozymes have proved useful for the identification of distinct regions of the proteins that are structurally and functionally important.

Introduction

Phosphatidylinositol-specific phospholipases C (PI-PLCs) comprise a diverse family of enzymes. They have been isolated from bacteria, protozoa, yeast, mold, plants, insects and mammals. Of the well characterized PI-PLCs, the bacterial enzymes are secreted while those of other organisms are intracellular enzymes. PI-PLCs catalyze the cleavage of the membrane lipid phosphatidylinositol (PI), or its phosphorylated derivatives, to produce diacylglycerol (DAG) and the water-soluble head group, phosphorylated myo-inositol. The smallest PI-PLCs, about 35 kDa in size, are produced by a variety of aerobic or anaerobic Gram-positive bacteria, including the pathogens Bacillus cereus, B. thuringiensis, Listeria monocytogenes, L. ivanovii, Staphylococcus aureus, Clostridium novyi and Rhodococcus equii. These enzymes are considered to be potential virulence factors [1], [2], but are also found in the non-pathogenic species L. seeligeri, Streptomyces antibioticus and the facultative anaerobe Gram-negative Cytophaga sp. In addition to being possible contributors to the pathogenicity of certain bacteria, the bacterial PI-PLCs are also an important diagnostic tool in the research on eukaryotic membrane proteins. Using their glycosyl-PI cleaving ability, proteins tethered to the cell membrane by glycosylphosphatidylinositol (GPI) anchors can be identified and released [3]. More recently, as a result of the crystal structure determination of bacterial PI-PLC and mammalian PI-PLCδ1, the single domain enzymes from bacteria have emerged as a useful model system for understanding the more complex and highly regulated second messenger-producing PI-PLC from eukaryotic cells (structural similarities between prokaryotic and eukaryotic PI-PLCs have been reviewed by Heinz et al. [4]; for a recent discussion of mammalian PI-PLCs see Katan [5]). Here, we discuss the biochemistry of bacterial PI-PLC, focusing on integrating the new structural and NMR data into current models of the action of these enzymes.

Section snippets

Reactions catalyzed and substrate specificity

PI-PLCs from B. cereus and B. thuringiensis have received the most attention. They have been shown to carry out two reactions (Fig. 1): The first step is a phosphotransferase reaction in which the phospholipid is cleaved, producing DAG and d-myo-inositol 1,2-cyclic phosphate, I(1,2)cP [6]. In a second reaction, the water-soluble intermediate product I(1,2)cP is hydrolyzed, yielding acyclic d-myo-inositol 1-phosphate, I(1)P. The second reaction proceeds at a rate that is about 10³-times slower

Sequence comparisons

The sequences of five bacterial PI-PLCs have been published: B. cereus, B. thuringiensis, L. monocytogenes, L. ivanovii and S. aureus. All are single polypeptide chains of similar lengths, about 300 amino acids. The highest degree of similarity is found among PI-PLCs from species of the same genus. Sequence identities are 69% for Listeria and 98% for Bacillus PI-PLCs. Only 22% or 24% identity exits between Listeria PI-PLC and the enzymes from Bacillus and S. aureus, respectively. With only

Resolution and assignment of all six histidines in B. cereus PI-PLC, and determination of pK_a values

The pK_a of the histidine imidazole group lies within the physiological pH range, and can therefore act as either a general acid or base. This versatility is one reason histidines play an important role in enzyme catalysis. Single peaks arising from the six histidines are observed in ¹H-¹³C heteronuclear single quantum coherence (HSQC) NMR experiments for B. cereus PI-PLC selectively labeled with ¹³C^ϵ1-histidine [29], [30]. The peaks have been assigned by preparing mutants in which five of the

Sequential S_N2 displacement

PI-PLC is a member of an important group of enzymes, the phosphodiesterases. These include deoxyribonucleases, ribonucleases, restriction endonucleases and the exonuclease activity of polymerases. Even ribozymes and macromolecular protein-RNA complexes such as spliceosomes have phosphodiesterase activities. The mechanisms of many of these enzymes have not been investigated though an in-line S_N2 displacement appears to be consistent with available data. In this mechanism, the attacking

Interfacial catalysis

A challenging aspect for studying the kinetics of phospholipases is that these lipolytic enzymes are water-soluble while their natural substrates are not soluble in water in the monomeric state but form a separate lipid phase. These enzymes, whether phospholipase C, D, or A₂, bind reversibly to phospholipid surfaces where they show interfacial activation; i.e., bound to the membrane surface, they cleave lipid substrates at a higher rate than would be possible with a monomeric substrate. Enzymes

Acknowledgements

We wish to thank our colleagues Drs. G. Bruce Birrell, Karen K. Hedberg and Dirk W. Heinz for useful discussions. This work was supported by NIH Grant 25698 from the General Medical Institute.

References (53)

S. Hirose et al.
Mammalian glycosylphosphatidylinositol-anchored proteins and intracellular precursors
Methods Enzymol.
(1995)
D.W. Heinz et al.
Structural and mechanistic comparison of prokaryotic and eukaryotic phosphoinositide-specific phospholipases C
J. Mol. Biol.
(1998)
M. Katan
Families of phosphoinositide-specific phospholipase C: structure and function
Biochim. Biophys. Acta
(1998)
K.S. Bruzik et al.
Toward the mechanism of phosphoinositide-specific phospholipases C
Bioorg. Med. Chem.
(1994)
J.J. Volwerk et al.
Functional characteristics of phosphatidylinositol-specific phospholipases C from Bacillus cereus and Bacillus thuringiensis
FEMS Microbiol. Lett.
(1989)
M.V. Ellis et al.
Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of PLCδ1
J. Biol. Chem.
(1998)
M.L. Guther et al.
The detection of phospholipase-resistant and -sensitive glycosyl-phosphatidylinositol membrane anchors by western blotting
Anal. Biochem.
(1994)
M.G. Low
The glycosyl-phosphatidylinositol anchor of membrane proteins
Biochim. Biophys. Acta
(1989)
J. Moser et al.
Crystal structure of the phosphatidylinositol-specific phospholipase C from the human pathogen Listeria monocytogenes
J. Mol. Biol.
(1997)
S. Stieger et al.
Glycosyl-phosphatidylinositol anchored acetylcholinesterase as substrate for phosphatidylinositol-specific phospholipase C from Bacillus cereus
Biochimie
(1991)

M.V. Ellis et al.

Structural requirements of phosphatidylinositol-specific phospholipase C δ1 for enzyme activity

Eur. J. Biochem.

(1993)

Cited by (102)

The Bacillus cereus group
2023, Molecular Medical Microbiology, Third Edition
The Bacillus cereus group, a group of highly phylogenetically related Bacillus species, includes the commonly studied species B. cereus, B. anthracis, and B. thuringiensis, as well as psychrotolerant (B. weihenstephanensis and B. wiedmannii), thermotolerant (B. cytotoxicus), morphologically distinct (B. mycoides and B. pseudomycoides) and probiotic (B. toyonensis) species. Nine novel Bacillus species have recently been isolated from marine sediments and seawater. The bacteria of this group, except B. anthracis, can cause systemic and local clinical cases, like pulmonary, eye, or wound infections. They are also notorious food pathogens causing diarrhea or vomiting. Emetic isolates usually represent a narrow lineage and synthesize thermostable plasmid-encoded toxin. The toxins nonhemolytic enterotoxin, hemolysin BL [a tripartite enterotoxin, consisting of a binding component (B) and two lytic components (L1 and L2)], and cytotoxin K have been rather well characterized. The global regulator PlcR has been intensively studied and unambiguously related to the regulation of toxins and multiple virulence factors. In recent years, the whole genome sequence of B. cereus strains has provided considerable information for genomic and functional studies. This chapter includes a survey of known virulence factors and outlines their potential or demonstrated contributions to virulence, in particular in relation to the group taxonomy.
An insight into bacterial phospholipase C classification and their translocation through Tat and Sec pathways: A data mining study
2019, Meta Gene
Bacterial phospholipases C (PLCs) are extra-cytoplasmic enzymes that play a crucial role in pathogenicity. Based on the substrates they hydrolyse, PLCs can be divided into four distinct groups viz. zinc-metallo, phosphatidylinositol-hydrolyzing, PLC from acid phosphatase superfamily and sphingomyelinase. PLCs are exported out of the cytoplasm to their functional locality either via Tat or Sec pathway. This study documents a sequence-based method for the classification of putative PLCs from different bacterial sources as well as prediction of their translocation pathways. The analysis indicates that PLCs belonging to a particular group show a distinct conservation of signature residues based on which classification could be done. Tat- and Sec-directed PLCs show differences in the characteristics of their signal peptide which include presence or absence of twin-arginine motif, signal peptide length and hydrophobicity. These features were used as markers for the assignment of translocation pathways. Majority of the PLCs are recognized as members of acid phosphatase superfamily and mostly show a tendency towards Tat pathway. Furthermore, this study confirms that apart from Sec-directed proteins, Tat-directed proteins also possess SecB-binding site implying the non-existence of any sequence-level distinction between Sec- and Tat-specific PLCs with respect to the number of potential SecB-binding site(s).
Phospholipid-Based Surfactants
2019, Biobased Surfactants: Synthesis, Properties, and Applications
In this chapter, in addition to a summary of updated understanding and progresses for biosynthesis (pathway), biological functions, and physicochemical properties of phospholipids (PLs), we review latest progresses in separation, analysis and characterization of PLs, strategy for chemo- or enzymatic modifications of PLs, particularly in the synthesis of lyso-PLs and structured PLs. Intensive discussion is given to the design and synthesis of functional PLs such as new phenophospholipids and applications of PLs as surfactants in different industrial sectors, such as food, pharma, and cosmetics.
Scrambling of natural and fluorescently tagged phosphatidylinositol by reconstituted G protein– coupled receptor and TMEM16 scramblases
2018, Journal of Biological Chemistry
Members of the G protein–coupled receptor and TMEM16 (transmembrane protein 16) protein families are phospholipid scramblases that facilitate rapid, bidirectional movement of phospholipids across a membrane bilayer in an ATP-independent manner. On reconstitution into large unilamellar vesicles, these proteins scramble more than 10,000 lipids/protein/s as measured with co-reconstituted fluorescent nitrobenzoxadiazole (NBD)-labeled phospholipids. Although NBD-labeled phospholipids are ubiquitously used as reporters of scramblase activity, it remains unclear whether the NBD modification influences the quantitative outcomes of the scramblase assay. We now report a refined biochemical approach for measuring the activity of scramblase proteins with radiolabeled natural phosphatidylinositol ([³H]PI) and exploiting the hydrolytic activity of bacterial PI-specific phospholipase C (PI-PLC) to detect the transbilayer movement of PI. PI-PLC rapidly hydrolyzed 50% of [³H]PI in large symmetric, unilamellar liposomes, corresponding to the lipid pool in the outer leaflet. On reconstitution of a crude preparation of yeast endoplasmic reticulum scramblase, purified bovine opsin, or purified Nectria haematococca TMEM16, the extent of [³H]PI hydrolysis increased, indicating that [³H]PI from the inner leaflet had been scrambled to the outer leaflet. Using transphosphatidylation, we synthesized acyl-NBD-PI and used it to compare our PI-PLC–based assay with conventional fluorescence-based methods. Our results revealed quantitative differences between the two assays that we attribute to the specific features of the assays themselves rather than to the nature of the phospholipid. In summary, we have developed an assay that measures scrambling of a chemically unmodified phospholipid by a reconstituted scramblase.
Differential subcellular distribution of four phospholipase C isoforms and secretion of GPI-PLC activity
2016, Biochimica et Biophysica Acta - Biomembranes
Citation Excerpt :
Enzymes of the phospholipase C (PLC) family can be found in pro- and eukaryotes [1,2].
Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms.
We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP₃. In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface.
Post-translational modifications of plant cell wall proteins and peptides: A survey from a proteomics point of view
2016, Biochimica et Biophysica Acta - Proteins and Proteomics
Citation Excerpt :
The GPI anchor provides stable attachment of the protein to the plasma membrane on its outer surface. The phosphatidylinositol moiety of the anchor can be cleaved by phosphatidylinositol-specific phospholipases C (PI-PLCs) [26]. Thus, GPI-anchored proteins may exist both at the plasma-membrane outer surface after cleavage and in a soluble form inside cell walls.
Plant cell wall proteins (CWPs) and peptides are important players in cell walls contributing to their assembly and their remodeling during development and in response to environmental constraints. Since the rise of proteomics technologies at the beginning of the 2000's, the knowledge of CWPs has greatly increased leading to the discovery of new CWP families and to the description of the cell wall proteomes of different organs of many plants. Conversely, cell wall peptidomics data are still lacking. In addition to the identification of CWPs and peptides by mass spectrometry (MS) and bioinformatics, proteomics has allowed to describe their post-translational modifications (PTMs). At present, the best known PTMs consist in proteolytic cleavage, N-glycosylation, hydroxylation of P residues into hydroxyproline residues (O), O-glycosylation and glypiation. In this review, the methods allowing the capture of the modified proteins based on the specific properties of their PTMs as well as the MS technologies used for their characterization are briefly described. A focus is done on proteolytic cleavage leading to protein maturation or release of signaling peptides and on O-glycosylation. Some new technologies, like top–down proteomics and terminomics, are described. They aim at a finer description of proteoforms resulting from PTMs or degradation mechanisms. This article is part of a Special Issue entitled: Plant Proteomics— a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

View all citing articles on Scopus

View full text

ReviewBacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids

Abstract

Introduction

Section snippets

Reactions catalyzed and substrate specificity

Sequence comparisons

Resolution and assignment of all six histidines in B. cereus PI-PLC, and determination of pKa values

Sequential SN2 displacement

Interfacial catalysis

Acknowledgements

Methods Enzymol.

J. Mol. Biol.

Biochim. Biophys. Acta

Bioorg. Med. Chem.

FEMS Microbiol. Lett.

J. Biol. Chem.

Anal. Biochem.

Biochim. Biophys. Acta

J. Mol. Biol.

Biochimie

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Biochim. Biophys. Acta

Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor?

Mol. Microbiol.

Cloning, expression, and mutagenesis of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: a potential staphylococcal virulence factor

Infect. Immun.

Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. I. purification, properties and phosphatase- releasing activity

Biochim. Biophys. Acta

Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: a 31P NMR study

Biochemistry

Substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus examined using the resolved enantiomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate)

Biochemistry

Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C

Biochemistry

Are d- and l-chiro-phosphoinositides substrates of phosphatidylinositol-specific phospholipase C?

Biochemistry

Phospholipids chiral at phosphorus. Stereochemical mechanism for the formation of inositol 1-phosphate catalyzed by phosphatidylinositol-specific phospholipase C

Biochemistry

The surface glycoconjugates of trypanosomatid parasites

Phil. Trans. R. Soc. Lond. B

Proteins on the surface of the malaria parasite and cell invasion

Parasitology

Primary structure of CD52

J. Biol. Chem.

Genetic diversity of Bacillus cereus/B. thuringiensis isolates from natural sources

Curr. Microbiol.

Structural requirements of phosphatidylinositol-specific phospholipase C δ1 for enzyme activity

Eur. J. Biochem.

Review
Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids

Resolution and assignment of all six histidines in B. cereus PI-PLC, and determination of pK_a values

Sequential S_N2 displacement

Phosphatidylinositol-specific phospholipase C from Bacillus cereus combines intrinsic phosphotransferase and cyclic phosphodiesterase activities: a ³¹P NMR study