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O R Veltman

Publications and source records attributed to O R Veltman.

15 recordsLinked to original sources
PubMed2001

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.

Amino Acid Substitution↗
PubMed1998

Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations.

The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallographic studies have shown that the active-site cleft is more closed in ligand-binding TLPs than in ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in "closure" and "opening" of the active-site cleft. Two hinge regions have been proposed. One is located around a conserved glycine 78; the second involves residues 135 and 136. The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly --> Ala mutations on catalytic activity. Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined. Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly. These mutants displayed a reduction in kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on kcat/Km for various substrates with effects on the Ki for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence kcat. The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.

Alanine↗
PubMed1998

A single calcium binding site is crucial for the calcium-dependent thermal stability of thermolysin-like proteases.

Thermostable thermolysin-like proteases (TLPs), such as the TLP of Bacillus stearothermophilus CU-21 (TLP-ste), bind calcium in one double (Ca1,2) and two single (Ca3, Ca4) calcium binding sites. The single sites are absent in thermolabile TLPs, suggesting that they are determinants of (variation in) TLP stability. Mutations in the Ca3 and Ca4 sites of TLP-ste indeed reduced thermal stability, but only mutations in the Ca3 site affected the calcium-dependence of stability. The predominant effect of the Ca3 site results from the fact that the Ca3 site is part of a region of TLP-ste, which unfolding is crucial for thermal inactivation. Thermal inactivation is not caused by the absence of calcium from the Ca3 site per se, but rather by unfolding of a region of TLP-ste for which stability depends on the occupancy of the Ca3 site. In accordance with this concept is the observation that the effects of mutations in the Ca3 site could be compensated by stabilizing mutations near this site. In addition, it was observed that the contribution of calcium binding to the Ca3 was substantially reduced in extremely stable TLP-ste variants containing multiple stabilizing mutations in the Ca3 region. Apparently, in these latter variants, unfolding of the Ca3 region contributes little to the overall process of thermal inactivation.

Amino Acid Sequence↗
PubMed1998

Engineering an enzyme to resist boiling.

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

Amino Acid Substitution↗
PubMed1998

Rendering one autolysis site in Bacillus subtilis neutral protease resistant to cleavage reveals a new fission.

Autolytic degradation of the thermolysin-like proteinase of Bacillus subtilis (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously we have reported five cleavage sites in TLP-sub [Van den Burg et al. (1990) Biochem. J. 272, 93-97]. In an attempt to render the enzyme less susceptible to autolytic breakdown, one of the fission sites, located between Leu-156 and Ile-157, was modified by replacing Ile-157, C-terminally located with respect to the fission site, by an Asp residue. Aspartic acid is less preferred at this position with respect to the substrate preference of TLP-sub. Modelling studies indicated that this mutation was unlikely to cause conformational changes in the enzyme. Although the 156-157 fission was not observed in the mutant enzyme, a new fission site, between Gly-148 and Val-149, was now observed.

Amino Acid Sequence↗
PubMed1997

Mutational analysis of a surface area that is critical for the thermal stability of thermolysin-like proteases.

Site-directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55-69 region of the thermolysin-like protease of Bacillus stearothermophilus (TLP-ste) to thermal stability. The importance of the 55-69 region was reflected by finding that almost all mutations had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutations affecting main chain flexibility, as well as by mutations affecting the interaction between the 55-69 region and the rest of the protease molecule. The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp59). In the case of the Asp57-->Ser mutation, the accompanying loss in stability was modest compared with the effects of other destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of a stabilizing salt bridge involving residues 65 and 11.

Amino Acid Sequence↗
PubMed1997

Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond.

The thermal inactivation of broad specificity proteases such as thermolysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis. Previous studies have revealed that a surface-located region in the N-terminal domain of the thermolysin-like protease produced by Bacillus stearothermophilus is crucial for thermal stability. In this region a disulfide bridge between residues 8 and 60 was designed by molecular modelling, and the corresponding single and double cysteine mutants were constructed. The disulfide bridge was spontaneously formed in vivo and resulted in a drastic stabilization of the enzyme. This stabilization presents one of the very few examples of successful stabilization of a broad specificity protease by a designed disulfide bond. We propose that the success of the present stabilization strategy is the result of the localization and mutation of an area of the molecule involved in the partial unfolding processes that determine thermal stability.

Disulfides↗
PubMed1997

Model building of a thermolysin-like protease by mutagenesis.

The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub). An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus thermoproteolyticus (thermolysin) and Bacillus cereus as templates. The largest portion of NP-sub could be modelled satisfactorily, using standard techniques, but several surface-located regions could only be modelled with a high degree of uncertainty. In order to make the model more reliable in these regions a 'model building by mutagenesis' approach was adopted. Mutations were designed such that their effect on thermal stability could indicate how their local environment should be modelled. This approach provided insight in the local structure of several regions in NP-sub that were hard to model on the basis of homology with the two known structures alone.

Amino Acid Sequence↗
PubMed1996

Analysis of structural determinants of the stability of thermolysin-like proteases by molecular modelling and site-directed mutagenesis.

The thermolysin-like protease (TLP) produced by Bacillus stearothermophilus CU21 (TLP-ste) differs at 43 positions from the more thermally stable thermolysin (containing 316 residues in total). Of these differences, 26 were analysed by studying the effect of replacing residues in TLP-ste by the corresponding residues in thermolysin. Several stabilizing mutations were identified but, remarkably, considerable destabilizing mutational effects were also found. A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertional difference between the two enzymes) appeared to make an important contribution to the stability of the enzyme. Mutations with large effects on stability were all localized in the beta-pleated N-terminal domain of TLP-ste, confirming observations that this domain has a lower intrinsic stability than the largely alpha-helical C-terminal domain. Rigidifying mutations such as Gly58-->Ala and Ala69-->Pro were among the most stabilizing ones. Apart from this observation, the analyses did not reveal general rules for stabilizing proteins. Instead, the results highlight the importance of context in evaluating the stability effects of mutations.

Amino Acid Sequence↗
PubMed1995

Structural determinants of the stability of thermolysin-like proteinases.

Thermolysin is a member of a family of homologous proteinases which differ in their resistance to thermally induced unfolding and subsequent autolytic degradation. Site-directed mutagenesis studies of the thermolysin-like proteinase (TLP) from Bacillus stearothermophilus (TLP-ste) show that its reduced resistance to thermally induced autolysis, as compared to thermolysin, is due to only some of the 44 naturally occurring amino-acid differences between them. In fact TLP-ste becomes more resistant than thermolysin by mutation of just a few of these amino-acids. The crucial differences are all localized to a solvent-exposed region in the N-terminal domain of TLP-ste.

Amino Acid Sequence↗
PubMed1993

Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines.

The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by proline. The mutations caused large positive (Ser-65-->Pro, Ala-69-->Pro) or negative (Thr-63-->Pro, Tyr-66-->Pro) changes in thermostability, which were explained on the basis of molecular modelling of the mutant proteins. The data show that the introduction of prolines at carefully selected positions in the protein can be a powerful method for stabilization.

Bacterial Proteins↗
PubMed1993

Comparison of different primer sets for detection of Chlamydia trachomatis by the polymerase chain reaction.

The sensitivity and specificity of the polymerase chain reaction (PCR) method was studied in vitro with HeLa cells infected with Chlamydia trachomatis serovar L2. Three different primer sets were studied; they were derived from the endogenous plasmid, the nonvariable part of the MOMP gene and the 16S ribosomal RNA (rRNA) gene. The plasmid primers were the most sensitive in the PCR method and detected at least 0.1 infectious unit of C. trachomatis in the presence of a superfluous amount of human DNA. Application of this plasmid PCR to 13 C. trachomatis culture-positive cervical smears containing < 10- > 200 inclusion-forming units showed that it was the most sensitive of the three methods and detected C. trachomatis in all samples. This correlates with the observation that the plasmid PCR method could detect C. trachomatis in cervical smears of four symptomatic patients for up to 3 weeks after the start of treatment with doxycycline. In contrast, the MOMP gene- and rRNA gene-directed PCR, as well as culture and direct immunofluorescence, gave negative results within 1 week. Therefore, we conclude that the plasmid primers are the best candidates for use in the PCR method in C. trachomatis screening programmes and clinical follow-up studies.

Bacterial Outer Membrane Proteins↗
PubMed1992

Introduction of a stabilizing 10 residue beta-hairpin in Bacillus subtilis neutral protease.

A 10 residue beta-hairpin, which is characteristic of thermostable Bacillus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully active after introduction of the loop. However, the mutant protein exhibited autocatalytic nicking and a 0.4 degree C decrease in thermostability. Two additional point mutations designed to improve the interactions between the enzyme surface and the introduced beta-hairpin resulted in reduced nicking and increased thermostability. After the introduction of both additional mutations in the loop-containing mutant, nicking was largely prevented and an increase in thermostability of 1.1 degrees C was achieved.

Amino Acid Sequence↗
PubMed1992

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease.

Cavities in the hydrophobic core of the neutral protease of Bacillus stearothermophilus were analyzed using a three-dimensional model that was inferred from the crystal structure of thermolysin, the highly homologous neutral protease of B. thermoproteolyticus (85% sequence identity). Site-directed mutagenesis was used to fill some of these cavities, thereby improving hydrophobic packing in the protein interior. The mutations had small effects on the thermostability, even after drastic changes, such as Leu284----Trp and Met168----Trp. The effects on T50, the temperature at which 50% of the enzyme is irreversibly inactivated in 30 min, ranged from 0.0 to +0.4 degrees C. These results can be explained by assuming that the mutations have positive and negative structural effects of approximately the same magnitude. Alternatively, it could be envisaged that the local unfolding steps, which render the enzyme susceptible towards autolysis and which are rate limiting in the process of thermal inactivation, are only slightly affected by alterations in the hydrophobic core.

Base Sequence↗