Metal ions and phosphatidylinositol 4,5-bisphosphate as interacting effectors of α-type plant phospholipase D.

Plant phospholipases D (PLD) are typically characterized by a C2 domain with at least two Ca binding sites. In vitro, the predominantly expressed α-type PLDs need 20-100 mM CaCl for optimum activity, whereas the essential activator of β- or γ-type PLDs, phosphatidylinositol 4,5-bisphosphate (PIP), plays a secondary role. In the present paper, we have studied the interplay between PIP and metal ion activation of the well-known α-type PLD from cabbage (PLDα).

Identification of a secretory phospholipase A2 from Papaver somniferum L. that transforms membrane phospholipids.

The full-length sequence of a new secretory phospholipase A2 was identified in opium poppy seedlings (Papaver somniferum L.). The cDNA of poppy phospholipase A2, denoted as pspla2, encodes a protein of 159 amino acids with a 31 amino acid long signal peptide at the N-terminus.

Purification, sequencing and characterization of phospholipase D from Indian mustard seeds.

Phospholipase D (PLD; E.C. 3.

Phospholipase A(2)-catalyzed acylation of lysophospholipids analyzed by experimental design.

The catalytic potential of phospholipase A2 (PLA2) for the synthesis of phospholipids with defined fatty acid structure in the sn-2 position has been underestimated hitherto because of very low conversion in most organic solvents. One of the most suitable solvents for PLA2-catalyzed phospholipid synthesis is glycerol. With the aim to analyze the effect of several interacting reaction parameters on the product yield, we studied the conversion of 1-palmitoyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PC) with oleic acid as model reaction in mixtures of glycerol and methanol or ethanol by methods of experimental design.

Probing selected structural regions in the secreted phospholipase A₂ from Arabidopsis thaliana for their impact on stability and activity.

In contrast to the well characterized secreted phospholipases A2 (sPLA2) from animals, their homologues from plants have been less explored. Their production in purified form is more difficult, and no data on their stability are known. In the present paper, different variants of the sPLA2 isoform α from Arabidopsis thaliana (AtPLA2α) were designed using a new homology model with the aim to probe the impact of regions that are assumed to be important for stability and catalysis.

Differences in the effect of phosphatidylinositol 4,5-bisphosphate on the hydrolytic and transphosphatidylation activities of membrane-bound phospholipase D from poppy seedlings.

The hydrolytic activity of phospholipase D (PLD) yielding phosphatidic acid from phosphatidylcholine and other glycerophospholipids is known to be involved in many cellular processes. In contrast, it is not clear whether the competitive transphosphatidylation activity of PLD catalyzing the head group exchange of phospholipids has a natural function. In poppy seedlings (Papaver somniferum L.

Discrimination between the regioisomeric 1,2- and 1,3-diacylglycerophosphocholines by phospholipases.

The artificial 1,3-diacyl-glycero-2-phosphocholines (1,3-PCs), which form similar aggregate structures as the naturally occurring 1,2-diacyl-sn-glycero-3-phosphocholines (1,2-PCs), were tested as substrates for different classes of phospholipases such as phospholipase A(2) (PLA(2)) from porcine pancreas, bee and snake venom, and Arabidopsis thaliana, phospholipase C (PLC) from Bacillus cereus, and phospholipase D (PLD) from cabbage and Streptomyces species. The regioisomers of the natural phospholipids were shown to bind to all investigated phospholipases with an affinity similar to the corresponding naturally occurring phospholipids, however their hydrolysis was reduced to different degrees (PLA(2)s and PLC) or even abolished (PLDs belonging to the PLD superfamily). The results are in accordance with binding models obtained by docking the substrates to the crystal structures or homology models of the phospholipases.

The transphosphatidylation potential of a membrane-bound phospholipase D from poppy seedlings.

Plant phospholipases D (PLDs) occur in a large variety of isoenzymes, which differ in Ca(2+) ion requirement, phosphatidylinositol-4,5-bisphosphate (PIP(2)) activation and substrate selectivity. In the present study a membrane-bound PLD has been identified in the microsomal fractions of poppy seedlings (Papaver somniferum). The maximum PLD activity is found after 2 days of germination in endosperms and after 3 days in developing seedlings.

Disulfide bonds of phospholipase A2 from bee venom yield discrete contributions to its conformational stability.

Disulfide bonds are known to be crucial for protein stability. To probe the contribution of each of the five disulfide bonds (C9-C31, C30-C70, C37-C63, C61-C95, and C105-C113) in bee venom phospholipase A(2) to stability, variants with deleted disulfide bonds were produced by substituting two serine residues for each pair of cysteine residues. The mutations started from the pseudo-wild-type variant (pWT) with the mutation I1A (Markert et al.

Refolding of the non-specific neutral protease from Bacillus stearothermophilus proceeds via an autoproteolytically sensitive intermediate.

A very thermostable variant of the thermolysin-like protease from Bacillus stearothermophilus (G8C/N60C) was previously created by introduction of a disulfide bond into the cysteine-free pseudo-wild type variant (pWT) and thus fixing the unfolding region 56-69. In the present paper, we show that G8C/N60C and pWT can be reactivated from the completely unfolded states, accessible at >or=7.5M guanidine hydrochloride, and analyze the kinetics of folding, autoproteolytic degradation and aggregation.

Plant phospholipases A2: perspectives on biotechnological applications.

The recent progress in knowledge on biochemical properties and functions of phospholipases A(2) in plants paved the way for approving the suitability of these enzymes for commercial use now. The secreted phospholipases A(2), representing one type of phospholipases A(2) occurring in plants, show distinct differences in substrate specificities with respect to headgroup and acyl chains of the glycerophospholipids in comparison to their counterparts from animal sources. The other type of phospholipases A(2) in plants, the patatin-related phospholipases A(2), is characterized by broad substrate specificity.

Modulation of phospholipase D activity in vitro.

Most phospholipases D (PLDs) occurring in microorganisms, plants and animals belong to a superfamily which is characterized by several conserved regions of amino acid sequence including the two HKD motifs necessary for catalytic activity. Most eukaryotic PLDs possess additional regulatory structures such as the Phox and Pleckstrin homology domains in mammalian PLDs and the C2 domain in most plant PLDs. Owing to recombinant expression techniques, an increasing number of PLDs from different organisms has been obtained in purified form, allowing the investigation of specific and unspecific interactions of the enzymes with regulatory components in vitro.

Secretory phospholipase A2-alpha from Arabidopsis thaliana: functional parameters and substrate preference.

The secretory phospholipase A2-alpha from Arabidopsis thaliana (AtsPLA2-alpha), being one of the first plant sPLA2s obtained in purified state, has been characterised with respect to substrate preference and optimum conditions of catalysis. The optima of pH, temperature, and calcium concentration were similar to the parameters of secretory PLA2s from animals. However, substrate preferences markedly differed.

Alpha-amylase from mung beans (Vigna radiata)--correlation of biochemical properties and tertiary structure by homology modelling.

Alpha-amylase from germinated mung beans (Vigna radiata) has been purified 600-fold to electrophoretic homogeneity and a final specific activity of 437 U/mg. SDS-PAGE of the final preparation revealed a single protein band of 46 kDa. The optimum pH was 5.

Production of synthetically created phospholipase A(2) variants with industrial impact.

Phospholipases A(2) (PLA(2)) play an important role for the production of lysophospholipids. Presently they are mainly obtained from porcine or bovine pancreas but these mammalian sources are not accepted in several fields of application. To make accessible a non-mammalian PLA(2) to industrial application, synthetic genes encoding PLA(2) from honey bee (Apis mellifera) with modified N-termini were constructed and expressed in Escherichia coli.

The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents.

Engineered extremely thermostable variants of the thermolysin-like protease from Bacillus stearothermophilus possessing an introduced disulfide bond G8C/N60C (double mutant, DM) and six additional amino acid substitutions in the exposed loop region 56-69 (Boilysin, BLN) have been probed with respect to stability toward water-miscible organic solvents and detergents. The solvent concentrations where 50% of enzyme activity were irreversibly lost (C(50)) decreased in the order methanol > 2-propanol > dimethylsulfoxide > dioxane > acetonitrile > dimethylformamide > acetone. The C(50) values were remarkably higher for the thermostable variants than for the wild-type enzymes.

Probing conserved amino acids in phospholipase D (Brassica oleracea var. capitata) for their importance in hydrolysis and transphosphatidylation activity.

In addition to hydrolysis of glycerophospholipids, phospholipases D (PLDs) catalyze the head group exchange. The molecular basis of this transphosphatidylation potential, which strongly varies for PLDs from different sources, is unknown hitherto. Recently, the genes of two PLD isoenzymes from white cabbage have been sequenced and expressed in Escherchia coli, yielding the basis for mutational studies.

Secretory phospholipase A2 from Arabidopsis thaliana: insights into the three-dimensional structure and the amino acids involved in catalysis.

A low-molecular weight phospholipase A2 from Arabidopsis thaliana, isoform phospholipase A2-alpha, has been expressed in Escherichia coli in the form of inclusion bodies, refolded, and purified to homogeneity to yield the active mature enzyme. The enzyme was characterized with respect to pH, temperature optimum, and Ca2+ ion requirement. The enzyme has been shown to be a true secretory phospholipase A2 that requires Ca2+ ions in the millimolar range and belongs to group XIB.

Two highly homologous phospholipase D isoenzymes from Papaver somniferum L. with different transphosphatidylation potential.

The genes of two phospholipase D (PLD) isoenzymes, PLD1 and PLD2, from poppy seedlings (2829 and 2828 bp) were completely sequenced. The two genes have 96.9% identity in the encoding region and can be assigned to the alpha-type of plant PLDs.

An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding.

The extreme thermal stabilization achieved by the introduction of a disulfide bond (G8C/N60C) into the cysteine-free wild-type-like mutant (pWT) of the neutral protease from Bacillus stearothermophilus[Mansfeld J, Vriend G, Dijkstra BW, Veltman OR, Van den Burg B, Venema G, Ulbrich-Hofmann R & Eijsink VG (1997) J Biol Chem272, 11152-11156] was attributed to the fixation of the loop region 56-69. In this study, the role of calcium ions in the guanidine hydrochloride (GdnHCl)-induced unfolding and autoproteolysis kinetics of pWT and G8C/N60C was analyzed by fluorescence spectroscopy, far-UV CD spectroscopy and SDS/PAGE. First-order rate constants (kobs) were evaluated by chevron plots (ln kobs vs.

The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus.

The thermolysin-like neutral protease from Bacillus stearothermophilus (TLP-ste) is usually produced extracellularly in Bacillus subtilis, where it is expressed as preproenzyme and subsequently processed in an autocatalytic, intramolecular process. To create the basis for the production of inactive mutants of TLP-ste, which cannot be processed in B. subtilis, we studied the expression of TLP-ste and its propeptide in cis and in trans in Escherichia coli.

Identification of phospholipase D from cabbage as N-terminally acetylated PLD2.

Recently, the genes of two isoenzymes of phospholipase D from white cabbage (PLD1 and PLD2) with molecular masses of 91.7 and 91.9 kDa, respectively, have been sequenced and expressed in Escherichia coli [Schäffner, I.

Boilysin and thermolysin in dipeptide synthesis: a comparative study.

Boilysin (BLN) is an engineered, highly thermostable neutral protease from Bacillus stearothermophilus. Its high resistance is based on the stabilization of a surface loop (amino acid residues 55-69) by eight amino acid exchanges, including the introduction of a disulphide bond. In the present study, BLN was compared with the well-known and structurally related thermolysin (from B.