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

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 (betaalpha)(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.     

Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean

Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian δ-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian δ-type PI-PLCs and yeast PI-PLC1, the putative Ca²⁺-binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane.     

Structure of the S. aureus PI-specific phospholipase C reveals modulation of active site access by a titratable π-cation latched loop

Staphylococcus aureus secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) as a virulence factor that is unusual in exhibiting higher activity at acidic pH values than other enzymes in this class. We have determined the crystal structure of this enzyme at pH 4.6 and pH 7.5. Under slightly basic conditions, the S. aureus PI-PLC structure closely follows the conformation of other bacterial PI-PLCs. However, when crystallized under acidic conditions, a large section of mobile loop at the αβ-barrel rim in the vicinity of the active site shows ~10 ? shift. This loop displacement at acidic pH is the result of a titratable intramolecular π-cation interaction between His258 and Phe249. This was verified by a structure of the mutant protein H258Y crystallized at pH 4.6, which does not exhibit the large loop shift. The intramolecular π-cation interaction for S. aureus PI-PLC provides an explanation for the activity of the enzyme at acid pH and also suggests how phosphatidylcholine, as a competitor for Phe249, may kinetically activate this enzyme.     

N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana

Phosphoinositide-specific phospholipase C's (PI-PLCs) are ubiquitous in eukaryotes, from plants to animals, and catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate into the two second messengers inositol 1,4,5-trisphosphate and diacylglycerol. In animals, four distinct subfamilies of PI-PLCs have been identified, and the three-dimensional structure of one rat isozyme, PLC-delta1, determined. Plants appear to contain only one gene family encoding PI-PLCs. The catalytic properties of plant PI-PLCs are very similar to those of animal enzymes. However, very little is known about the regulation of plant PI-PLCs. All plant PI-PLCs comprise three domains, X, Y and C2, which are also conserved in isoforms from animals and yeast. We here show that one PI-PLC isozyme from Arabidopsis thaliana, AtPLC2, is predominantly localized in the plasma membrane, and that the conserved N-terminal domain may represent an EF-hand domain that is required for catalytic activity but not for lipid binding.     

Expression, characterization, and crystallization of the catalytic core of rat phosphatidylinositide-specific phospholipase C delta 1.

Phosphatidylinositide-specific phospholipase Cs (PI-PLCs) catalyze the calcium-dependent hydrolysis of phosphatidylinositides in response to diverse stimuli in higher eukaryotes. Mammalian PI-PLCs contain divergent regulatory regions, but all share three conserved regions: an N-terminal pleckstrin homology (PH) domain, X, and Y. We report the high-level expression and characterization of a recombinant "catalytic core" of rat PI-PLC delta 1 that contains the catalytically essential X and Y regions, but not the PH domain. The expressed protein, PI-PLC delta delta 1-134, is catalytically active versus phosphatidylinositol 4,5-bisphosphate in deoxycholate micelles with a K(m) of 182 microM and a Vmax of 27 mumol/min/mg. PI-PLC delta delta 1-134 is monomeric and monodisperse as judged by dynamic light scattering. Far-UV CD indicates a structure with approximately 35% alpha-helix. A reversible change in the near-UV CD spectrum is observed on addition of calcium, suggesting that calcium can bind PI-PLC delta delta 1-134 in the absence of phospholipid. Triclinic crystals of PI-PLC delta delta 1-134 have been obtained that diffract beyond 2.4 A resolution under cryogenic conditions. Based on Vm = 2.72 Da/A3 and on the self-rotation function, there are two PI-PLC delta delta 1-134 molecules per asymmetric unit that are related to each other by a noncrystallographic axis of approximate twofold symmetry parallel to a.     

Engineering a catalytic metal binding site into a calcium-independent phosphatidylinositol-specific phospholipase C leads to enhanced stereoselectivity

Eukaryotic phosphatidylinositol-specific phospholipase Cs (PI-PLCs) utilize calcium as a cofactor during catalysis, whereas prokaryotic PI-PLCs use a spatially conserved guanidinium group from Arg69. In this study, we aimed to construct a metal-dependent mutant of a bacterial PI-PLC and characterize the catalytic role of the metal ion, seeking an enhanced understanding of the functional differences between these two positively charged moieties. The following results indicate that a bona fide catalytic metal binding site was created by the single arginine-to-aspartate mutation at position 69: (1) The R69D mutant was activated by Ca(2+), and the activation was specific for R69D, not for other mutants at this position. (2) Titration of R69D with Ca(2+), monitored by (15)N-(1)H HSQC (heteronuclear single quantum coherence) NMR, showed that addition of Ca(2+) to R69D restores the environment of the catalytic site analogous to that attained by the WT enzyme. (3) Upon Ca(2+) activation, the thio effect of the S(P)-isomer of the phosphorothioate analogue (k(O)/k(Sp) = 4.4 x 10(5)) approached a value similar to that of the WT enzyme, suggesting a structural and functional similarity between the two positively charged moieties (Arg69 and Asp69-Ca(2+)). The R(P)-thio effect (k(O)/k(Rp) = 9.4) is smaller than that of the WT enzyme by a factor of 5. Consequently, R69D-Ca(2+) displays higher stereoselectivity (k(Rp)/k(Sp) = 47,000) than WT (k(Rp)/k(Sp) = 7600). (4) Results from additional mutagenesis analyses suggest that the Ca(2+) binding site is comprised of side chains from Asp33, Asp67, Asp69, and Glu117. Our studies provide new insight into the mechanism of metal-dependent and metal-independent PI-PLCs.     

Competition between Anion Binding and Dimerization Modulates Staphylococcus aureus Phosphatidylinositol-specific Phospholipase C Enzymatic Activity

Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) is a secreted virulence factor for this pathogenic bacterium. A novel crystal structure shows that this PI-PLC can form a dimer via helix B, a structural feature present in all secreted, bacterial PI-PLCs that is important for membrane binding. Despite the small size of this interface, it is critical for optimal enzyme activity. Kinetic evidence, increased enzyme specific activity with increasing enzyme concentration, supports a mechanism where the PI-PLC dimerization is enhanced in membranes containing phosphatidylcholine (PC). Mutagenesis of key residues confirm that the zwitterionic phospholipid acts not by specific binding to the protein, but rather by reducing anionic lipid interactions with a cationic pocket on the surface of the S. aureus enzyme that stabilizes monomeric protein. Despite its structural and sequence similarity to PI-PLCs from other Gram-positive pathogenic bacteria, S. aureus PI-PLC appears to have a unique mechanism where enzyme activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane.     

The activity of phosphoinositide-specific phospholipase C is required for vegetative growth and cell wall regeneration in Coprinopsis cinerea

Three isotypes of phosphoinositide-specific phospholipase C designated CcPLC1, CcPLC2, and CcPLC3 were identified in Coprinopsis cinerea, through a search of the genome sequence database. The functional role of the PI-PLCs were studied by using U73122, which specifically inhibits the activity of PI-PLC. The specificity of the inhibitor effect was confirmed by using an inactive structural analog U73433. The inhibition of PI-PLCs activity resulted in severely retarded germination of basidiospores and oidia, reduced hyphal growth, knobbly hyphal tips with many irregular side branches, and aberrant (branch-like structure) clamp cells. Furthermore, U73122 definitely inhibited cell wall formation. Here we report that PI-PLCs play important roles in various aspects of C. cinerea biology.     

Cloning, tissue distribution and sub-cellular localisation of phospholipase C X-domain containing protein (PLCXD) isoforms

Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes comprise a small family of receptor-regulated phosphodiesterases that control many cellular processes by the regulation of cytosolic calcium and/or the activity of several protein kinases. To date, six distinct classes of PI-PLC are known to exist in mammals. Here we characterise a seventh class of PI-PLC, which contains only the catalytic X domain in its structure, termed phospholipase C X-domain containing protein (PLCXD). At least three tissue-specific PLCXD isoforms exist in humans, comprising hPLCXD-1, hPLCXD-2 and hPLCXD-3, with hPLCXD-2 exhibiting three C-terminal spliceforms (2.1, 2.2 and 2.3). Specific amino acids known to be essential for the catalytic function of PI-PLCs were found to be conserved in all three human PLCXDs and over-expression of hPLCXD-1, 2.1 and 3 in the HeLa cell line increased endogenous PI-PLC activity. Human PLCXD isoforms exhibited tissue-specific expression profiles in mice and humans and immunocytochemistry revealed distinct sub-cellular localisations when over-expressed in human cultured cell lines. These novel proteins may therefore possess fundamental, and as yet uncharacterised roles in cell physiology.     

Characterization of phosphatidylinositol-specific phospholipase C (PI-PLC) from Lilium daviddi pollen

The phosphatidylinositol-specific phospholipase C (PI-PLC) activity is detected in purified Lilium pollen protoplasts. Two PI-PLC full length cDNAs, LdPLC1 and LdPLC2, were isolated from pollen of Lilium daviddi. The amino acid sequences for the two PI-PLCs deduced from the two cDNA sequences contain X, Y catalytic motifs and C2 domains. Blast analysis shows that LdPLCs have 60-65% identities to the PI-PLCs from other plant species. Both recombinant PI-PLCs proteins expressed in E. coli cells show the PIP(2)-hydrolyzing activity. The RT-PCR analysis shows that both of them are expressed in pollen grains, whereas expression level of LdPLC2 is induced in germinating pollen. The exogenous purified calmodulin (CaM) is able to stimulate the activity of the PI-PLC when it is added into the pollen protoplast medium, while anti-CaM antibody suppresses the stimulation effect caused by exogenous CaM. PI-PLC activity is enhanced by G protein agonist cholera toxin and decreased by G protein antagonist pertussis toxin. Increasing in PI-PLC activity caused by exogenous purified CaM is also inhibited by pertussis toxin. A PI-PLC inhibitor, U-73122, inhibited the stimulation of PI-PLC activity caused by cholera toxin and it also leads to the decrease of [Ca(2+)](cyt) in pollen grains. Those results suggest that the PPI-PLC signaling pathway is present in Lilium daviddi pollen, and PI-PLC activity might be regulated by a heterotrimeric G protein and extracellular CaM.     

AtPLC2, a gene encoding phosphoinositide-specific phospholipase C,is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana

A cDNA encoding a phosphoinositide-specific phospholipase C (PI-PLC) from the higher plant Arabidopsis thaliana was cloned and characterized.The gene corresponding to this cDNA is designated AtPLC2. The overall structure of the predicted AtPLC2 protein is similar to those of plant PI-PLCs and mammalian -type PI-PLCs. Northern blot analysis revealed that AtPLC2 is expressed constitutively whereas AtPLC1S, another gene for PI-PLC of Arabidopsis, is induced by environmental stresses such as dehydration and salinity, indicating that the function of AtPLC2 is distinct from that of AtPLC1S. The AtPLC2 mRNA was detected in vegetative and floral tissues. We determined the positions of these two PI-PLCs genes on Arabidopsis chromosomes by RFLP mapping using P1 genomic clones.     

Isolation of cDNAs encoding typical and novel types of phosphoinositide-specific phospholipase C from the moss Physcomitrella patens

Two cDNAs encoding proteins, PpPLC1 and PpPLC2, with catalytic and C2 domains conserved in plant phosphoinositide-specific phospholipase C (PI-PLC) were isolated from Physcomitrella patens. The N domain, which has been identified in Arabidopsis PI-PLCs as an EF hand-like domain, was found in both isoforms, although that in PpPLC2 was a split type. At micromolar Ca2+ concentrations, PpPLC1 preferentially hydrolysed phosphatidylinositol-4,5-bisphosphate, while PpPLC2 showed no specificity. Furthermore, at millimolar Ca2+, phosphatidylinositol was hydrolysed by PpPLC2, but not by PpPLC1. Thus, PpPLC1 and PpPLC2 are typical and novel types of plant PI-PLC, respectively.     

Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca(2+)-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P). Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP. Cosolvents [isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)] also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles. Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates. PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the T(m) for PI-PLC thermal denaturation decreased linearly with added iPrOH). However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein. At 30% iPrOH and 4 degrees C (well below the T(m) for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal. iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate. This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little effect on protein secondary structure.     

A Ribokinase Family Conserved Monovalent Cation Binding Site Enhances the MgATP-induced Inhibition in E.?coli Phosphofructokinase-2

The enzymatic activity of secreted phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes is associated with bacterial virulence. Although the PI-PLC active site has no obvious lid, molecular-dynamics simulations suggest that correlated loop motions may limit access to the active site, and two Pro residues, Pro²⁴⁵ and Pro²⁵⁴, are associated with these correlated motions. Whereas the region containing both Pro residues is quite variable among PI-PLCs, it shows high conservation in virulence-associated, secreted PI-PLCs that bind to the surface of cells. These regions of the protein are also associated with phosphatidylcholine binding, which enhances PI-PLC activity. In silico mutagenesis of Pro²⁴⁵ disrupts correlated motions between the two halves of Bacillus thuringiensis PI-PLC, and Pro²⁴⁵ variants show significantly reduced enzymatic activity in all assay systems. PC still enhanced activity, but not to the level of wild-type enzyme. Mutagenesis of Pro²⁵⁴ appears to stiffen the PI-PLC structure, but experimental mutations had minor effects on activity and membrane binding. With the exception of P245Y, reduced activity was not associated with reduced membrane affinity. This combination of simulations and experiments suggests that correlated motions between the two halves of PI-PLC may be more important for enzymatic activity than for vesicle binding.     

Phosphatidylinositol-specific phospholipase C of Bacillus cereus: cloning, sequencing, and relationship to other phospholipases

The phosphatidylinositol (PI)-specific phospholipase C (PLC) of Bacillus cereus was cloned into Escherichia coli by using monoclonal antibody probes raised against the purified protein. The enzyme is specific for hydrolysis of the membrane lipid PI and PI-glycan-containing membrane anchors, which are important structural components of one class of membrane proteins. The protein expressed in E. coli comigrated with B. cereus PI-PLC in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as detected by immunoblotting, and conferred PI-PLC activity on the host. This enzyme activity was inhibited by PI-PLC-specific monoclonal antibodies. The nucleotide sequence of the PI-PLC gene suggests that this secreted bacterial protein is synthesized as a larger precursor with a 31-amino-acid N-terminal extension to the mature enzyme of 298 amino acids. From analysis of coding and flanking sequences of the gene, we conclude that the PI-PLC gene does not reside next to the gene cluster of the other two secreted phospholipases C on the bacterial chromosome. The deduced amino acid sequence of the B. cereus PI-PLC contains a stretch of significant similarity to the glycosylphosphatidylinositol-specific PLC of Trypanosoma brucei. The conserved peptide is proposed to play a role in the function of these enzymes.     

Inhibition of phosphatidylinositol-specific phospholipase C: Studies on synthetic substrates,inhibitors and a synthetic enzyme

Enzyme inhibition studies on phosphatidylinositol-specific phospholipase C (PI-PLC) from B. Cereus were performed in order to gain an understanding of the mechanism of the PI-PLC family of enzymes and to aid inhibitor design. Inhibition studies on two synthetic cyclic phosphonate analogues (1,2) of inositol cyclic-1:2-monophosphate (cIP), glycerol-2-phosphate and vanadate were performed using natural phosphatidylinositol (PI) substrate in Triton X100 co-micelles and an NMR assay. Further inhibition studies on PI-PLC from B. Cereus were performed using a chromogenic, synthetic PI analogue (DPG-PI), an HPLC assay and Aerosol-OT (AOT), phytic acid and vanadate as inhibitors. For purposes of comparison, a model PI-PLC enzyme system was developed employing a synthetic Cu(II)-metallomicelle and a further synthetic PI analogue (IPP-PI). The studies employing natural PI substrate in Triton X100 co-micelles and synthetic DPG-PI in the absence of surfactant indicate three classes of PI-PLC inhibitors: (1) active-site directed inhibitors (e.g. 1,2); (2) water-soluble polyanions (e.g. tetravanadate, phytic acid); (3) surfactant anions (e.g. AOT). Three modes of molecular recognition are indicated to be important: (1) active site molecular recognition; (2) recognition at an anion-recognition site which may be the active site, and; (3) interfacial (or hydrophobic) recognition which may be exploited to increase affinity for the anion-recognition site in anionic surfactants such as AOT. The most potent inhibition of PI-PLC was observed by tetravanadate and AOT. The metallomicelle model system was observed to mimic PI-PLC in reproducing transesterification of the PI analogue substrate to yield cIP as product and in showing inhibition by phytic acid and AOT.     

Phosphoinositide-specific phospholipase C: structural basis for catalysis and regulatory interactions

Phosphoinositide-specific phospholipase C (PI-PLC) isozymes have an important role in cellular responses to a variety of extracellular signals. Recently, the three-dimensional structures of their isolated domains and of the multidomain core, common to all PI-PLCs, have been solved. This provided an insight into the domain organization of PI-PLCs and, together with the structure-function analysis, contributed towards an understanding of the molecular mechanisms of catalysis and regulation.     

Density-dependent inhibition of cell growth is correlated with the activity of a cell surface phosphatidylinositol-specific phospholipase C.

We previously identified a novel phospatidylinositol-specific phospholipase C (PI-PLC) present at the surface of Swiss 3T3 cells using a fluorescent analog of PI and showed that this cell surface PI-PLC (csPI-PLC) activity increases with increasing cell density (J. Biol. Chem. 265, 5337-5340 (1990)). Here, we find that csPI-PLC activity also increased in cultures in which growth was inhibited due to serum deprivation. csPI-PLC was inhibited by agents that inhibit other mammalian PI-PLCs, but not by treatments which inhibit glycosyl PI-PLCs (GPI-PLCs). We also extended our studies using fluorescent PI to other cell types and found that csPI-PLC activity was present only in cell lines that exhibit growth inhibition upon reaching confluency (Swiss 3T3, 3T3-L1, BALB/c 3T3, and normal rat kidney (NRK) fibroblasts), but not in cell lines that are tumorigenic and/or do not exhibit growth inhibition in a density-dependent manner (Chinese hamster ovary (CHO), mouse L, SV-40 transformed BALB/c 3T3 (SV-T2), baby hamster kidney (BHK), and Chinese hamster lung (V79) fibroblasts). These results support the hypothesis that csPI-PLC plays a role in the density-dependent inhibition of cell growth.     

Al(3+)-mediated changes on membrane fluidity affects the activity of PI-PLC but not of PLC

We investigated whether Al(3+)-mediated changes in membrane fluidity can affect the activity of prokaryotic enzymes phospholipase C (PLC) and phospholipase C-phosphatidyl inositol specific (PI-PLC) in liposomes of phosphatidyl choline (PC), PC:phosphatidyl inositol (PI), or PC and polyphosphoinositides (PPI). Al(3+) (10-100 microM) promoted membrane rigidification, evaluated with the probes 1,6-diphenyl-1,3,5-hexatriene and Laurdan, and followed the order: PC:PPI>PC:PI>PC. Al(3+) (25 and 50 microM) did not affect PLC-mediated hydrolysis of PC, PI and PIP(2), but stimulated PIP hydrolysis (48.6%). PI-PLC did not affect PC, PI, and PIP concentrations, but caused a 67% decrease in PIP(2). Al(3+) significantly inhibited PIP(2) hydrolysis in a concentration-dependent (25-50 microM) manner. Results suggest that the inhibition of PIP(2) hydrolysis by Al(3+) could be partially due to a higher lipid packing induced by Al(3+) which could affect the interaction between the enzyme and its substrate.