Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in discrete steps: (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. Water-soluble cIP was used as the substrate to study the cyclic phosphodiesterase activity and interfacial behavior of PI-PLC. Different detergent micelles and phospholipid vesicles were used to examine if "interfacial activation" of the enzyme could occur toward a soluble substrate. Almost all detergents examined activated the enzyme at least 2-fold, with PC species yielding the largest increases in PI-PLC specific activity. Kinetic parameters were measured in the absence and presence of several representative detergents (e.g., Triton X-100 and diheptanoylphosphatidylcholine (diC7PC)). Gel filtration experiments showed that, under these conditions, the cIP did not partition to any measurable extent with these detergent micelles. The concentration at which half the maximum activation was observed occurred near the detergent CMC. Both Km and Vmax were altered by the presence of a surface: Km decreased to different degrees depending on the detergent, while Vmax increased substantially. The Km for cIP was 90 mM without detergent and decreased to 29 mM with diC7PC micelles added; Vmax increased almost 7-fold in the presence of diC7PC micelles. The enzyme efficiency (Vmax/Km) in the presence of diC7PC increased more than 21-fold, but it was still 20-fold lower than initial phosphotransferase activity for monomeric dihexanoylphosphatidylinositol. The poor efficiency of the cyclic phosphodiesterase activity is largely due to substrate binding affinity. The dependence of rate on substrate concentration exhibits cooperative behavior, especially without detergent. This cooperativity is discussed in terms of protein aggregation and ligand binding sites on the enzyme.
Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits 'interfacial activation' toward the water-soluble substrate myo-inositol 1,2-(cyclic)phosphate [Zhou et al. (1997) Biochemistry 36, 347-355]. The activation of PI-PLC enzyme is optimal with PC or PE interfaces. NMR experiments (TRNOE and 31P line width analyses) were carried out to investigate the interaction of PI-PLC with activator amphiphiles. These studies showed that the enzyme had high affinity for phosphatidylcholine (or PE) molecules with dissociation constants of 0.5 and 0.3 mM for diC6PC and diC7PC, respectively. TRNOE cross-peaks of bound PC were confirmed to represent intramolecular relaxation pathways using partially perdeuterated PC molecules consistent with a single molecule binding tightly. The large activation by a PC interface can be explained by a single PC molecule binding specifically to PI-PLC and anchoring the enzyme-lipid complex to the interface. Other interfaces, such as micellar diC8PS, can activate PI-PLC about 2-3-fold; however, the monomers of these detergents showed little affinity for the enzyme as measured by TRNOE or 31P NMR line widths. The 3.6-fold activation produced by polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (compared to the 15-fold activation generated by nonpolymerized PC vesicles) was comparable to the nonspecific activation of other detergents. This confirmed that single-PC molecule binding was allosteric and anchored the enzyme in the interface. The conformation of interfacially activated enzyme is discussed in term of the stabilization of a critical surface loop and helix B observed with weak intensity in the X-ray crystal structure.
A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.
A series of symmetric short-chain phosphatidylinositols (PI), including dihexanoyl-PI, diheptanoyl-PI (racemic as well as D and L forms), and 2-methoxy inositol-substituted diheptanoyl-PI, have been synthesized, characterized, and used to investigate key mechanistic questions about phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis. Key results include the following: (i) bacterial PI-PLC exhibits a 5-6-fold "interfacial activation" when its substrate is present in an interface as opposed to existing as a monomer in solution (in fact, the similarity to the activation observed with nonspecific PLC enzymes suggests a similarity in activation mechanisms); (ii) the 2-OH must be free since the enzyme cannot hydrolyze diheptanoyl-2-O-methyl-PI (this is most consistent with the formation of inositol cyclic 1,2-phosphate as a necessary step in catalysis); (iii) the inositol ring must have the D stereochemistry (the L-inositol attached to the lipid moiety is neither a substrate nor an inhibitor); and (iv) the presence of noninhibitory L-PI with the D-PI substrate relieves the diacylglycerol product inhibition detected at approximately 30% hydrolysis.
During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P 2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P 2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P 2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P 2 synthesis.
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