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H M Verheij

Publications and source records attributed to H M Verheij.

At least 37 records · Page 2Linked to original sources
PubMed1998

Ammodytin L, an inactive phospholipase A2 homologue with myotoxicity in mice, binds to the presynaptic acceptor of the beta-neurotoxic ammodytoxin C in Torpedo: an indication for a phospholipase A2 activity-independent mechanism of action of beta-neurotoxins in fish?

A Ser48 phospholipase A2-homologue, ammodytin L, which is myotoxic in mammals and devoid of any phospholipase A2 activity, completely inhibits the specific binding of the neurotoxic phospholipase A2, ammodytoxin C, to fish presynaptic membranes from Torpedo marmorata electric organ. In cross-linking experiments, 125I-ammodytin L labels the same membrane proteins as 125I-ammodytoxin C (70, 38.5-57.4 and 19.7 kDa). The formation of these adducts is completely prevented by the presence of ammodytoxin C but not of a non-toxic phospholipase A2, ammodytin I2. A chimeric phospholipase A2, constructed by associating the N-terminal half of ammodytoxin to the C-terminal half of ammodytin L, possesses a low, but significant phospholipase A2 activity, however it is not toxic to mice, probably due to abolition of the specific neuronal acceptor binding in mammals. Nevertheless, the chimeric phospholipase A2 is able to interact with the ammodytoxin acceptor in Torpedo marmorata electric organ. The existence of neuronal acceptors for ammodytin L and for the chimeric phospholipase A2 suggests that they may act as neurotoxins in fish. As ammodytin L does not possess any enzymatic activity it, therefore, appears to be an excellent tool to investigate the mechanism of action of beta-neurotoxins independently of their phospholipase A2 activity.

Amino Acid Sequence↗
PubMed1998

Substrate specificity of Staphylococcus hyicus lipase and Staphylococcus aureus lipase as studied by in vivo chimeragenesis.

Staphylococcus hyicus lipase (SHL) and Staphylococcus aureus lipase (SAL) are highly homologous enzymes, yet they show remarkable differences in their biochemical characteristics. SHL displays a high phospholipase activity, hydrolyses neutral lipids, and has no chain length preference, whereas SAL only degrades short-chain fatty acid esters. To identify the regions in the primary sequence of SHL responsible for phospholipase activity and chain length selectivity, a set of histidine-tagged SAL/SHL chimeras was generated by in vivo recombination in Escherichia coli. Several classes of chimeric enzymes were identified on the basis of restriction site analysis. All chimeras were well-expressed as active enzymes. They were characterized for their specific activities on both phospholipids and p-nitrophenyl esters of various chain lengths. Phospholipase activity appeared to be determined by three regions, all located in the C-terminal domain of SHL. Testing of the enzymatic activity of the chimeras toward p-nitrophenyl esters showed that chain length selectivity is defined by elements within the region of residues 180-253. Moreover, also residues along the stretch 275-358 contribute to the binding of acyl chains. Interestingly, several chimeras were even more active than the parent enzymes on long-chain p-nitrophenyl esters.

Amino Acid Sequence↗
PubMed1998

A 7.4-A projection structure of outer membrane phospholipase A from Escherichia coli by electron crystallography.

Outer membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Escherichia coli. Two-dimensional crystals of OMPLA were grown by reconstitution of purified protein into lipid bilayers via detergent dialysis and were studied by electron crystallography. A 7.4-A projection map reveals OMPLA molecules exhibiting an oval-shaped domain of 30 x 20 A resembling the beta-barrel structure characteristic of porins, which is associated with a 25-A elongated domain of lower density.

Bacterial Outer Membrane Proteins↗
PubMed1998

Staphylococcal lipases: molecular characterisation, secretion, and processing.

Up to date five different staphylococcal lipase genes, two of Staphylococcus aureus (sal-1 and sal-2), two of Staphylococcus epidermidis (sel-1 and sel-2) and one of Staphylococcus hyicus (sh1) have been cloned and sequenced. All corresponding proteins are organised as pre-pro-enzymes: the pre-region represents the signal peptide, the pro-region has a length between 207 and 267 amino acids, and the mature part comprises 380 to 400 amino acids. We found that the lipases are secreted in the pro-lipase form. The processing of the pro-form to the mature enzyme occurs extracellular by a specific protease. Interestingly the pro-lipase reveals not much less activity compared to the mature lipase. There are evidences that the pro-region acts as an intramolecular chaperone which facilitates translocation not only of the native lipase but also of a number of completely unrelated proteins fused to the pro-peptide. It was also observed that the pro-region protects the proteins from proteolytic degradation. While the Staphylococcus aureus and Staphylococcus epidermidis lipases have only lipase (esterase) activity, the related Staphylococcus hyicus enzyme (SHL) is distinguished by both lipase and phospho-lipase activity. The biochemical and catalytic properties of these lipases are described in the accompanying article (Simons, J.W., Götz, F., Egmont, M.R. and Verheij, H.M., 1998. Staphylococcal lipases: Biochemical properties. Accompanying article).

Bacterial Proteins↗
PubMed1998

Biochemical properties of staphylococcal (phospho)lipases.

Various staphylococci secrete lipases which require calcium ions for activity, and have profound preferences for substrates with different chain lengths. The lipase from Staphylococcus hyicus is exceptional since it has higher phospholipase than lipase activity. This paper gives an overview of the biochemical properties of these enzymes. It appears that chain length selectivity of these enzymes resides in the acylation step. Interfaces mainly influence the acylation step. Calcium ions do not influence the rate of acylation or deacylation although stabilise the enzyme against denaturation. Molecular modelling based on the X-ray structure of Pseudomonas glumae lipase was used to construct a model of the staphylococcal lipases. With this model the position of serveral residues involved in stubstrate selectivity was predicted. Moreover, a sequence element could be assigned that may function as the so-called lid domain in staphylococcal lipases. Sequence alignment of four staphylococcal lipases, and lipases from P. glumae and Bacillus thermocatenulatus identified several potential calcium ligands, one of which was verified by site directed mutagensesis. It is concluded that stabilisation of lipases by calcium ions might be a more general phenomenon than recognized so far.

Amino Acid Sequence↗
PubMed1998

The phospholipase activity of Staphylococcus hyicus lipase strongly depends on a single Ser to Val mutation.

Site-directed mutagenesis and domain exchange were used to investigate the role of the C-terminal domains of Staphylococcus hyicus lipase (SHL) and S. aureus lipase (SAL) in substrate selectivity. The introduction of a single point mutation coding for the substitution of Val for Ser356 in SHL yields an enzyme which has retained full lipase activity, but with more than 12-fold lower phospholipase activity. Starting with this S356V variant of SHL the C-terminal 40 amino acids were replaced by the corresponding SAL sequence. Although 23 amino acid changes were introduced simultaneously the impact on the phospholipase/lipase activity ratio was only 4-fold. We therefore conclude that in the C-terminal domain it is Ser356 which mainly determines phospholipase activity. The introduction of a Val357 to Ser substitution in SAL did not turn SAL into a phospholipase, showing that residues from other domains contribute to this activity as well. The results are discussed in view of the sequence homology of lipases and (lyso)phospholipases.

Amino Acid Sequence↗
PubMed1998

Cutinase binding and activity at the triolein-water interface monitored by oil drop tensiometry.

Changes of the oil-water interfacial tension resulting from binding of Fusarium solani pisi cutinase and subsequent lipid hydrolysis were investigated using the oil drop technique. An ELISA was developed to determine the amount of cutinase bound to the triolein-water interface after biotinylation of the enzyme. Cutinase irreversibly adsorbs to a maximum value of about 2 mg/m2. A minimal specific activity of 110 mumol/min/mg was calculated for cutinase acting on a single oil droplet, which is close to the activity found for triglyceride emulsions. At a maximum surface load cutinase could generate one monolayer of fatty acid products per second at the interface. It was found that oleic acid rapidly dissolves into the oil phase under the conditions used. The interfacial tension measured reflects the adsorption of cutinase to the oil droplet and also responds to the fate of the hydrolysis products. A model is presented that describes the catalytic events at the oil-water interface during lipid hydrolysis.

Adsorption↗
PubMed1997

Dissecting the catalytic mechanism of staphylococcal lipases using carbamate substrates: chain length selectivity, interfacial activation, and cofactor dependence.

p-Nitrophenyl N-alkylcarbamates with different alkyl chains were used as substrates to determine separately the carbamylation and decarbamylation rates of the lipases from Staphylococcus hyicus and S. aureus. Both enzymes are reversibly inhibited by these compounds due to a rapid carbamylation of their active site serines followed by a slow decarbamylation. The carbamylation reaction is strongly pH-dependent and the pH profile suggests that an unprotonated histidine is required for this reaction. In contrast, the decarbamylation is pH-independent suggesting the presence of a hydrogen bond between the active site histidine and the carbamyl moiety. S. hyicus lipase preferably reacts with medium to long chain carbamates with an optimum for eight carbon atoms. In contrast, S. aureus lipase is highly specific for short chain carbamates. These results are in agreement with the respective substrate preferences of both lipases toward natural lipids. The decarbamylation rates of both enzymes hardly depend on the alkyl chain length, and from this it is concluded that chain length selectivity is expressed in the first step of catalysis. Both the carbamylation and decarbamylation reaction rates of S. hyicus lipase are enhanced in the presence of micelles, the activation effect being most pronounced in the first step. For the S. aureus lipase only a small influence of interfaces on both reaction steps was observed. These results are discussed in view of a possible role of a lid covering the active site. Kinetic experiments in the presence and absence of calcium strongly suggest that calcium ions are important for the structural stabilization of the unmodified as well as of the carbamylated enzymes. This structural function of calcium was supported by urea unfolding experiments, from which it appeared that for both enzymes the free energy for unfolding is significantly lower in the absence of calcium. In conclusion our results show that kinetic differences between both lipases reside in the acylation step, and that calcium is important for the structural stabilization of the unmodified, and moreover, the acylated enzymes.

Carbamates↗
PubMed1997

An affinity column for phospholipase A2 based on immobilised acylaminophospholipid analogues.

A synthetic route was developed to prepare 2-acylamino phospholipid analogues suitable for immobilisation. The inhibitors, synthesised in either the (R)- and (S)-configuration, carried an omega-carboxyl group in one acyl chain for immobilisation to the matrix. As a matrix Sepharose 6B, derivatised with a polar, non-charged 16 atom spacer was used. Low-molecular weight phospholipase A2 binds in a calcium-dependent way to the immobilised (S)-inhibitor and not to the immobilised (R)-inhibitor which shows that binding involves specific active site interactions rather than hydrophobic chromatography. The specificity was further demonstrated by the fact that the immobilised (S)-inhibitor binds porcine pancreatic and snake venom phospholipases A2, but not the porcine pancreatic zymogen. Moreover, a mutant porcine pancreatic phospholipase A2 in which the active side residue His48 has been replaced by Gln, was not bound by the column. This column material might be applicable for affinity purification of phospholipase A2 and for screening of phage display libraries.

Acylation↗
PubMed1997

Dimerization regulates the enzymatic activity of Escherichia coli outer membrane phospholipase A.

The outer membrane phospholipase A (OMPLA) of Escherichia coli is present in a dormant state in the cell envelope. The enzyme is activated by various processes, which have in common that they perturb the outer membrane. Kinetic experiments, chemical cross-linking, and analytical ultracentrifugation were carried out with purified, detergent-solubilized OMPLA to understand the underlying mechanism that results in activation. Under conditions in which the enzyme displayed full activity, OMPLA was dimeric. High detergent concentrations or very dilute protein concentrations resulted in low specific activity of the enzyme, and under those conditions the enzyme was monomeric. The cofactor Ca2+ was required for dimerization. Covalent modification of the active site serine with hexadecylsulfonylfluoride resulted in stabilization of the dimeric form and a loss of the absolute calcium requirement for dimerization. The results of these experiments provide evidence for dimerization as the molecular mechanism by which the enzymatic activity of OMPLA is regulated. This dimerization probably plays a role in vivo as well. Data from chemical cross-linking on whole cells indicate that OMPLA is present in the outer membrane as a monomer and that activation of the enzyme induces dimerization concurrent with the appearance of enzymatic activity.

Detergents↗
PubMed1997

Crystal structure of cutinase covalently inhibited by a triglyceride analogue.

Cutinase from Fusarium solani is a lipolytic enzyme that hydrolyses triglycerides efficiently. All the inhibited forms of lipolytic enzymes described so far are based on the use of small organophosphate and organophosphonate inhibitors, which bear little resemblance to a natural triglyceride substrate. In this article we describe the crystal structure of cutinase covalently inhibited by (R)-1,2-dibutyl-carbamoylglycero-3-O-p-nitrophenylbutyl-phos phonate, a triglyceride analogue mimicking the first tetrahedral intermediate along the reaction pathway. The structure, which has been solved at 2.3 A, reveals that in both the protein molecules of the asymmetric unit the inhibitor is almost completely embedded in the active site crevice. The overall shape of the inhibitor is that of a fork: the two dibutyl-carbamoyl chains point towards the surface of the protein, whereas the butyl chain bound to the phosphorous atom is roughly perpendicular to the sn-1 and sn-2 chains. The sn-3 chain is accommodated in a rather small pocket at the bottom of the active site crevice, thus providing a structural explanation for the preference of cutinase for short acyl chain substrates.

Carboxylic Ester Hydrolases↗
PubMed1997

Topology of the outer membrane phospholipase A of Salmonella typhimurium.

The outer membrane phospholipase A (OMPLA) of Enterobacteriaceae has been proposed to span the membrane 14 times as antiparallel amphipathic beta-strands, thereby exposing seven loops to the cell surface. We have employed the epitope insertion method to probe the topology of OMPLA of Salmonella typhimurium. First, missense mutations were introduced at various positions in the pldA gene, encoding OMPLA, to create unique BamHI sites. These BamHI sites were subsequently used to insert linkers, encoding a 16-amino-acid B-cell epitope. Proper assembly of all mutant proteins was revealed by their heat modifiability in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The accessibility of the inserted epitopes was assessed. Immunofluorescence analysis of intact cells with antibodies against the inserted epitope showed that three of seven predicted loops are indeed cell surface exposed. Trypsin accessibility experiments verified the cell surface exposure of two additional loops and provided support for the proposed periplasmic localization of three predicted turns. For two other predicted exposed loops, the results were not conclusive. These results support to a large extent the proposed topology model of OMPLA. Furthermore, the observation that the substitutions Glu66Pro and Glu247Gly virtually abolished enzymatic activity indicates that these residues might play a major role in catalysis.

Amino Acid Sequence↗
PubMed1996

Action of cutinase at the triolein-water interface. Characterisation of interfacial effects during lipid hydrolysis using the oil-drop tensiometer as a tool to study lipase kinetics.

Interfacial events during lipid hydrolysis by cutinase are described as measured with the oil-drop tensiometer. A linear relation between enzyme concentration and initial decrease of oil-water interface tension (gamma o/w) due to lipolytic activity was observed. The amount of hydrolysis products showed a non-linear relation with gamma o/w. Hydrolysis is linear with time, even when the area occupied by the fatty acid molecules exceeds the drop surface by a factor 7000. At pH 9.0, fatty acids were found to partition mainly in the oil phase. Formation of calcium soaps and ionization increase the impact of fatty acids on gamma o/w without affecting enzyme activity. The presence of fatty acids at the interface, added prior to cutinase, delayed hydrolysis effects on gamma o/w. Fatty acids in the water phase almost completely abolished adsorption effects on gamma o/w, when the concentration was over the critical micellar concentration (cmc).

Biophysical Phenomena↗
PubMed1996

The lipase from Staphylococcus aureus. Expression in Escherichia coli, large-scale purification and comparison of substrate specificity to Staphylococcus hyicus lipase.

The genes coding for the mature part of the lipases from Staphylococcus aureus NCTC8530 and Staphylococcus hyicus have been cloned and overexpressed in Escherichia coli as fusion proteins with an N-terminal hexa-histidine tag. The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus lipase and 13 mg/g for S. hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with full retention of activity of S. aureus lipase. Whereas S. hyicus lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium- and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases.

Esters↗
PubMed1996

Escherichia coli outer membrane phospholipase A: role of two serines in enzymatic activity.

In the outer membrane phospholipase A (OMPLA) of Escherichia coli, Ser144 has previously been identified by chemical modification as the active site serine residue. In a specific OMPLA-negative mutant strain, the pldA gene coding for OMPLA was shown to differ from the wild-type gene by a single point mutation, resulting in the substitution of Ser152 by phenylalanine. The role in catalysis of these two serine residues in OMPLA was investigated by site-directed mutagenesis. Ser144 and Ser152 were replaced one at the time by either alanine, valine, phenylalanine, threonine, or cysteine. Ser152 was furthermore replaced by asparagine. Replacement of Ser144 by cysteine resulted in 1% residual activity, whereas the other substitutions at this position yielded virtually inactive enzymes. Substitution of Ser 152 by threonine or asparagine resulted in 40% and 2% residual activity respectively, whereas all other substitutions at this position resulted in the loss of enzymatic activity. We propose that Ser144 is the nucleophile in catalysis, and that Ser152 is involved in hydrogen bonding either to the catalytic triad or in the oxyanion hole.

Amino Acid Sequence↗
PubMed1996

Targeting of porcine pancreatic phospholipase A2 to human platelets. Introduction of an RGD sequence and acyl-group by chemical modification.

In the present study we prepared by chemical modification a series of porcine pancreatic phospholipase A2 (PLA) derivatives, that bind to the activated glycoprotein (GP) IIb/IIIa complex and hydrolyse phospholipids in the outer leaflet of the platelet membrane. To the native enzyme, an RGD-containing peptide was coupled to introduce affinity for GPIIb/IIIa in combination with lauric acid to improve binding to the membrane. As controls, derivatives containing only one of these modifications were prepared. Acylation of the enzyme improved the affinity for densely packed phospholipids, as deduced by kinetic analyses. After stimulation of platelets, the RGD-containing PLAs bound to GPIIb/IIIa since GRGDS peptide and a monoclonal antibody against the complex interfered with binding. No binding was found with native PLA. The binding seen with lauric acid PLA was not mediated by GPIIb/IIIa. All modified PLAs induced 1-3% hydrolysis of [3H]arachidonic-acid-labelled phospholipids in resting platelets. After activation with alpha-thrombin, hydrolysis increased to 17%, corresponding to about 90% of [3H]arachidonate-labelled phospholipids in the outer leaflet of the plasma membrane. RGD-containing PLAs were more active than lauroyl PLA, and their activity was mediated via GPIIb/IIIa since GRGDS inhibited release of [3H]arachidonic acid. Acylation of the RGD-containing PLAs did not further improve the hydrolytic properties. We conclude that chemical modification of PLA leads to a targetted hydrolytic action and could be a basis for the design of enzymes that specifically destroy activated platelets.

Acylation↗
PubMed1996

The anticoagulant effect of the human secretory phospholipase A2 on blood plasma and on a cell-free system is due to a phospholipid-independent mechanism of action involving the inhibition of factor Va.

Blood platelets play a central role in haemostasis by leading to plug formation and by increasing the efficiency of blood coagulation. We have previously shown that blood platelets contain a group II secretory phospholipase A2 (sPLA2 grII) which is released into the extracellular medium upon activation but is unable to stimulate blood platelets. We presently reported an investigation of the putative involvement of the human sPLA2 grII (hsPLA2 grII) in the coagulation process, both in the absence and in the presence of activated platelets. We show that this enzyme prolongs the recalcification time of blood plasma even in the presence of activated platelets. The positive action of blood platelets on coagulation is correlated, at least in part, with the appearance at the cellular surface of procoagulant phospholipids which constitute a potential target for hsPLA2 grII. We therefore investigated the involvement of its enzymatic activity in the anticoagulant effect of this enzyme. We observed that the replacement of CaCl2 by SrCl2 to initiate the coagulation cascade did not suppress, but rather increased, the inhibitory action of hsPLA2 grII. Moreover, hsPLA2 grII hydrolyzed only a minor proportion of platelet phospholipids, and it did not affect plasma phospholipids. Taken together, these observations strongly suggest that the major action of hsPLA2 grII on blood coagulation does not involve the hydrolysis of phospholipids, in contrast with the strong anticoagulant effect of the group II venom phospholipase A2 from Crotalus durrissus terrificus. We next studied which step of the coagulation cascade was affected by hsPLA2 grII. Using purified coagulation factors, we demonstrated that hsPLA2 grII strongly inhibited the prothrombinase activity. This inhibitory effect was independent of the presence of phospholipids but required factor Va, leading to the hypothesis that hsPLA2 grII inhibited this factor. Further, the anticoagulant effect of hsPLA2 grII was observed on normal and factor-X-deficient plasma, but not on factor-V-deficient plasma. In conclusion, the anticoagulant action of hsPLA2 grII is based on a nonenzymatic mechanism of action involving the inhibition of factor Va.

Animals↗