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Renate Ulbrich-Hofmann

Publications and source records attributed to Renate Ulbrich-Hofmann.

At least 19 recordsLinked to original sources
PubMed2007

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. Therefore, the stabilization of this loop region also protects the molecule from irreversible inactivation by solvents, and inactivation seems to follow principally the same mechanism as thermal inactivation. However, in contrast to thermal inactivation where the corresponding T(50) values of DM and BLN differed by 10 K, the differences of the C(50) values of DM and BLN were not significant. Detergents had great effects on proteolytic activities which were dependent on the individual detergent and its concentration, but mostly without significant differences between the enzyme variants. These effects were inactivating (SDS, sulfobetaine) or strongly activating (CTAB, CHAPS). Triton X-100 and Tween 20 were activating or inactivating at low and high concentrations, respectively. In all detergents, stabilities of the enzymes were strongly decreased. However, the more thermostable variants were affected by the detergents to the same extent as the wild-type enzymes suggesting that the mechanism of detergent inactivation is different from that of thermal inactivation.

Amino Acid Sequence↗
PubMed2006

Natural and engineered ribonucleases as potential cancer therapeutics.

By reason of their cytotoxicity, ribonucleases (RNases) are potential anti-tumor drugs. Particularly members from the RNase A and RNase T1 superfamilies have shown promising results. Among these enzymes, Onconase, an RNase from the Northern Leopard frog, is furthest along in clinical trials. A general model for the mechanism of the cytotoxic action of RNases includes the interaction of the enzyme with the cellular membrane, internalization, translocation to the cytosol, and degradation of ribonucleic acid. The interplay of these processes as well as the role of the thermodynamic and proteolytic stability, the catalytic activity, and the capability of the RNase to evade the intracellular RNase inhibitor has not yet been fully elucidated. This paper discusses the various approaches to exploit RNases as cytotoxic agents.

Animals↗
PubMed2006

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. In the present paper, three sequence characteristics of the isoenzyme (PLD2) that corresponds to the often used enzyme isolated from cabbage leaves have been probed for their importance in hydrolysis as well as transphosphatidylation activities: (i) the two HKD motifs, (ii) the C terminus and (iii) the eight cysteine residues. All these regions or amino acids are highly conserved in alpha-type plant PLDs. Based on multiple alignments, predictions of secondary structure and comparisons of hydrophobicity profiles, 35 enzyme variants were created and assayed. All positions tested proved to be very sensitive towards amino acid exchanges with respect to hydrolytic activity in the absence of glycerol as well as to the ratio of hydrolytic and transphosphatidylation activities in the presence of glycerol. A significant increase of total activity and transphosphatidylation activity could be obtained by the substitutions C310S and C625S.

Amino Acid Sequence↗
PubMed2006

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. On the basis of the three-dimensional structures of secretory phospholipase A2 forms (sPLA2s) from bee venom and bovine pancreas, a homology model was generated. Analysis of this model and alignments of different plant sPLA2s showed that the common His-Asp dyad of animal sPLA2s does not exist in plant sPLA2s. In place of the aspartate residue of the dyad, the plant enzymes of group XIA contain a histidine residue, and the enzymes of group XIB contain a serine or an asparagine residue. Mutagenesis of amino acids supposed to be involved in catalysis has shown that His62, the calcium-coordinating Asp63, and the above-mentioned Ser79 residue are essential for activity.

Amino Acid Sequence↗
PubMed2006

Contribution of structural peculiarities of onconase to its high stability and folding kinetics.

Onconase (ONC) from Rana pipiens is the smallest member of the ribonuclease A (RNase A) superfamily. Despite a tertiary structure similar to RNase A, ONC is distinguished by an extremely high thermodynamic stability. In the present paper we have probed the significance of three structural regions, which exhibit structural peculiarities in comparison to RNase A, for the stability of ONC to temperature and guanidine hydrochloride induced denaturation: (i) the N-terminal pyroglutamate residue, (ii) the hydrophobic cluster between helix I and the first beta-sheet, and (iii) the C-terminal disulfide bond. For this purpose, the enzyme variants <E1E-, <E1P-, F28T-, F28A-, F36Y-, and C87A/C104A-ONC were produced and studied in equilibrium and kinetic measurements. The destabilizing influence of the mutations strongly depended on the modified structural region. The exchanges of the N-terminal pyroglutamate (<E1E- and <E1P-ONC) had the smallest impact (DeltaDeltaG([D])50% = 4.2 and 7.0 kJ mol(-)(1)), while interferences in the hydrophobic cluster (F28T-, F28A-, and F36Y-ONC) had larger effects (DeltaDeltaG([D])50% = 22.2, 20.9, and 19.5 kJ mol(-)(1)). The removal of the C-terminal disulfide bond (C87A/C104A-ONC) showed the largest influence on stability (DeltaDeltaG([D])50% = 32.0 kJ mol(-)(1)). As concluded from the comparison of DeltaDeltaG([D])50% and DeltaDeltaG++(U)[D]50%, all destabilization effects were exclusively caused by increased unfolding rate constants except for C87A/C104A-ONC, where unfolding as well as folding was impacted. Of all amino acid residues investigated, Phe28, which is unique for ONC among the ribonucleases, had the greatest importance for rate of unfolding. Our data on the folding and unfolding kinetics indicate that the strong stabilization of ONC in comparison to RNase A is caused by a dramatic deceleration of the unfolding reaction.

Amino Acid Substitution↗
PubMed2006

Inhibition of pancreatic ribonuclease A aggregation by antibodies raised against the native enzyme and its N-terminal dodecapeptide.

Pancreatic ribonuclease A (RNase A) has been shown to aggregate moderately and gradually at 65 degrees C. Antibodies raised against the dodecapeptide KETAAAKFERQG corresponding to the N-terminal 1-12 amino acid residues of RNase A (Npep) as well as native RNase A were effective in lowering RNase A aggregation at 65 degrees C. The antiRNase A antibodies were, however, more protective. The binding of antiNpep antibodies to the N-terminal region of RNase A may interfere with initiation of oligomerization of the enzyme and consequently its aggregation. The antiRNase A antibodies were presumably more effective in protecting RNase A against aggregation by binding to multiple epitopes of the enzyme including the N-terminal region and hence restricting the interaction of the monomers.

Animals↗
PubMed2005

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. The corresponding amino acid sequences do not contain any signal sequences. One Asn-glycosylation site, six and two phosphorylation sites for protein kinase C and tyrosine kinase, respectively, and two phosphatidylinositol-4,5-bisphosphate binding motifs could be identified. Like in most plant PLDs, two HKD motifs and one C2 domain are present. PLD1 and PLD2 have ten and nine cysteine residues. The two enzymes were expressed in E. coli and purified to homogeneity by Ca2+ ion-mediated hydrophobic interaction chromatography. The Ca2+ ion concentration needed for carrier binding of the two enzymes in chromatography as well as for optimum activity was found to be considerably higher (>100 mM) than with other alpha-type plant PLDs. Although PLD1 and PLD2 differ in eleven amino acids only, they showed remarkable differences in their transphosphatidylation activity. Two amino acid exchanges within and near the first HKD motif contribute to this difference as shown by the A349E/E352Q-variant of PLD2.

Amino Acid Motifs↗
PubMed2005

Phospholipase D and its application in biocatalysis.

Phospholipase D (PLD) from plants or microorganisms is used as biocatalyst in the transformation of phospholipids and phospholipid analogs in both laboratory and industrial scale. In recent years the elucidation of the primary structure of many PLDs from several sources, as well as the resolution of the first crystal structure of a microbial PLD, have yielded new insights into the structural basis and the catalytic mechanism of this catalyst. This review summarizes some new results of PLD research in the light of application.

Catalysis↗
PubMed2005

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. In contrast to thermolysin, subtilisin and alpha-lytic protease, which could be obtained only in the presence of the corresponding propeptides, TLP-ste could be produced as an active mature enzyme in E. coli in the absence of its prosequence. Surprisingly, however, a much more effective access to active mature protease was found when TLP-ste (devoid of its prosequence) was expressed as protein with an N-terminal His6 tag which accumulated in the form of inclusion bodies. Completely unexpected, the protein could be renatured from the inclusion bodies after solubilization in guanidine hydrochloride solutions in high yields. Purification to homogeneity was possible by affinity chromatography on Bacitracin silica as well as by immobilized metal ion affinity chromatography. By addition of separately expressed propeptide to the renaturation mixture yields of renaturation could not be increased significantly, confirming that the propeptide is not essential for proper folding of the enzyme or its stabilization during the folding process. Also in vivo, the expression levels of active mature TLP-ste in Escherichia coli did not significantly differ when the mature sequence was expressed alone or coexpressed with the prosequence in cis or in trans.

Anti-Bacterial Agents↗
PubMed2005

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. GdnHCl concentration). The kobs of unfolding showed a difference of nearly six orders of magnitude (DeltaDeltaG# = 33.5 kJ.mol(-1) at 25 degrees C) between calcium saturation (at 100 mM CaCl2) and complete removal of calcium ions (in the presence of 100 mM EDTA). Analysis of the protease variant W55F indicated that calcium binding-site III, situated in the critical region 56-69, determines the stability at calcium ion concentrations between 5 and 50 mM. In the chevron plots the disulfide bridge in G8C/N60C shows a similar effect compared with pWT as the addition of calcium ions, suggesting that the introduced disulfide bridge fixes the region (near calcium binding-site III) that is responsible for unfolding and subsequent autoproteolysis. Owing to the presence of the disulfide bridge, the DeltaDeltaG# is 13.2 kJ.mol(-1) at 25 degrees C and 5 mM CaCl2. Non-linear chevron plots reveal an intermediate in unfolding probably caused by local unfolding of the loop 56-69. The occurrence of this intermediate is prevented by calcium concentrations of > 5 mM, or the introduction of the disulfide bridge G8C/N60C.

2-Propanol↗
PubMed2004

Mass spectrometric characterization of peptides derived by peptic cleavage of bovine beta-casein.

This study investigated the digestion of the milk protein beta-casein with pepsin under gastro-analogous conditions. Peptide sequences were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with post-source decay as well as liquid chromatography-tandem mass spectrometry by means of database searching. The new software tool, Mascot Distiller, improved the identification rate remarkably. In the case of small peptides, such as di- and tri-peptides, which are promising candidates for intestinal absorption and possible biological effects, identification was possible only after spectrum simulation and manual matching. A list of 41 identified peptides having 2-36 amino acids is given, and unexpected cleavage sites for pepsin are reported. Sequence coverage was 75%.

Amino Acid Sequence↗
PubMed2004

Behaviour of a recombinant cabbage (Brassica oleracea) phospholipase D immobilized on CNBr-activated and antibody supports.

Recombinant cabbage (Brassica oleracea) PLD2 (phospholipase D2) immobilized covalently on CNBr-activated Sepharose expressed low activity (approximately 10%), while that immobilized by binding on to anti-PLD2 IgG-Sepharose was more active (approximately 38%). Coupling of PLD2 to CNBr-activated Sepharose resulted in significant improvement in storage stability without affecting its thermostability, as compared with the soluble enzyme. Binding of PLD2 to the antibody support, however, rendered the enzyme remarkably labile to high temperatures and storage.

Animals↗
PubMed2004

Probing the unfolding region of ribonuclease A by site-directed mutagenesis.

Ribonuclease A contains two exposed loop regions, around Ala20 and Asn34. Only the loop around Ala20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacent beta-sheet around Thr45) is the first region of the ribonuclease A molecule that becomes susceptible to thermolysin and trypsin under unfolding conditions. This second region therefore has been suggested to be involved in early steps of unfolding and was designated as the unfolding region of the ribonuclease A molecule. Consequently, modifications in this region should have a great impact on the unfolding and, thus, on the thermodynamic stability. Also, if the Ala20 loop contributes to the stability of the ribonuclease A molecule, rigidification of this flexible region should stabilize the entire protein molecule. We substituted several residues in both regions without any dramatic effects on the native conformation and catalytic activity. As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/R33S/F46Y. The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in DeltaG can be mainly attributed to an increased unfolding rate. Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule.

Amino Acid Substitution↗
PubMed2003

Protein prosthesis: a nonnatural residue accelerates folding and increases stability.

Nonnatural residues can endow proteins with desirable properties. Here, replacing a proline residue that has a cis peptide bond in native ribonuclease A with 5,5-dimethyl-l-proline is shown to accelerate protein folding by 6-fold and enhance conformational stability by DeltaTm = 2.8 +/- 0.3 degrees C while having no effect on enzymatic activity. The rational use of this and other prosthetic segments could enable chemotherapeutic proteins to survive longer in vivo or retain activity after oral administration.

Kinetics↗
PubMed2003

Two uncommon phospholipase D isoenzymes from poppy seedlings (Papaver somniferum L.).

Phospholipase D (PLD) has been detected in seedlings of Papaver somniferum L. cv. Lazúr (Papaveraceae). Purification of the enzyme revealed the existence of two forms of PLD (named as PLD-A and PLD-B). The two enzymes strongly differ in their catalytic properties. The pH optima were found at pH 8.0 for PLD-A and at pH 5.5 for PLD-B. While both enzymes show hydrolytic activity toward phosphatidylcholine (PC) and phosphatidyl-p-nitrophenol (PpNP), PLD-B only was able to catalyze the exchange of choline in PC by glycerol. Both enzymes were activated by Ca(2+) ions with an optimum concentration of 10 mM. In contrast to PLDs from other plants, PLD-B was still more activated by Zn(2+) ions with an optimum concentration of 5 mM. The apparent molecular masses of PLD-A and PLD-B, derived from sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), were estimated to be 116.4 and 114.1 kDa. N-terminal protein sequencing indicated N-terminal blockage in both cases. The isoelectric points were found to be 8.7 for PLD-A and 6.7 for PLD-B. Both enzymes were shown to be N-linked glycoproteins. This paper is the first report on PLD in poppy and indicates some important differences of the two enzyme forms to other PLDs known so far.

Calcium Chloride↗
PubMed2003

Proline versus charge concept for protein stabilization against proteolytic attack.

The virtue of the so-called 'proline concept' and the 'charge concept' for stabilizing protease-susceptible regions of a protein structure was compared on bovine pancreatic ribonuclease A. Alanine 20 and serine 21, both of which are located in a loop that is susceptible to the unspecific proteases subtilisin Carlsberg, subtilisin BPN', proteinase K and elastase, were replaced with proline or lysine by site-directed mutagenesis. The rate constant of proteolysis was decreased by up to three orders of magnitude for the proline mutants depending on the site of the mutation and the protease used. In contrast, substitution by lysine increased the proteolytic resistance by only one order of magnitude characterizing the 'proline concept' as superior to the 'charge concept'. Although the four applied proteases are considered to be unspecific, the degree of stabilization of the ribonuclease molecule varied considerably, indicating the impact of individual differences in their substrate specificity on the proteolytic resistance and degradation pathway of the target protein.

Amino Acid Substitution↗
PubMed2002

Activity of phospholipase C in two-phase systems.

Although phospholipase C (PLC) is known to be activated by water-insoluble organic solvents, most activity assays have been designed to work in an aqueous milieu. Here a sensitive method is described for the determination of PLC activity in two-phase systems. The assay is based on the hydrolysis of phosphatidylcholine (PC) in chloroform/buffer. The initial rates of the reaction are determined by densitometric quantification of the product 1,2-diacylglycerol after its separation by high-performance TLC and staining with a CuSO4/H3PO4 or p-methoxybenzaldehyde/H2SO4 reagent. The method is examined for the determination of Vmax and Km values of PCs with varying length acyl chains (C10-C18). The comparison of the kinetic parameters with the Vmax and Km values of the same substrates in the conventional titrimetric assay, using sodium deoxycholate for micellization of PC, demonstrates the high efficiency of PLC in the two-phase emulsion system.

Acylation↗