Contribution of the C-terminal amino acid to the stability of <i>Bacillus subtilis</i> neutral protease

Eijsink, Vincent G. H.; Vriend, Gerrit; Burg, Bertus van den; Venema, Gerard; Stulp, Ben K.

doi:10.1093/protein/4.1.99

Cited by 39 publications

(28 citation statements)

References 20 publications

(26 reference statements)

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“…5; Table 3 Examination of the three residues in a three-dimensional model of the B. stearothermophilus Npr, constructed on the basis of the well-defined thermolysin structure (6), showed that residues 4, 59, and 66 are located at the surface of the protein (Fig. 7).…”

Section: Discussionmentioning

confidence: 99%

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product

Burg¹,

Enequist²,

Haar³

et al. 1991

J Bacteriol

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By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolyticaily active phages on plates supplemented with 0.8% skim milk, chromosomal B. caldolyticus DNA fragments that specified proteolytic activity were obtained. Subcloning of one of these fragments in a protease-deficient Bacillus subtUis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb Hinfl-MluI fragment contained an open reading frame (ORF) that specified a protein of 544 amino acids. This ORF was denoted as the B. caldolyticus npr gene, because the nucleotide and amino acid sequences of the ORF were highly similar to that of the Bacillus stearothermophilus npr gene. Additionally, the size, pH optimum, and sensitivity to the specific Npr inhibitor phosphoramidon of the secreted enzyme indicated that the B. caldolyticus enzyme was a neutral protease. The B. stearothermophilus and B. caldolyticus enzymes differed at only three amino acid positions. Nevertheless, the thermostability and optimum temperature of the B. caldolyticus enzyme were 7 to 8°C higher than those of the B. stearothermophilus enzyme. In a three-dimensional model of the B. stearothermophilus Npr the three substitutions (Ala-4 to Thr, Thr-59 to Ala, and Thr-66 to Phe) were present at solvent-exposed positions. The role of these residues in thermostabiity was analyzed by using site-directed mutagenesis. It was shown that all three amino acid substitutions contributed to the observed difference in thermostability between the neutral proteases from B. stearothermophilus and B.caldolyticus.Bacterial species belonging to the genus Bacillus secrete a variety of enzymes, some of which are of economical importance, in particular the ax-amylases and proteinases (5). The various Bacillus species show large variations in optimum growth temperatures, which are often, but not invariably, reflected in the thermostabilities of their extracellular enzymes (23). By comparing thermostable and thermolabile variants of homologous enzymes, information on the mechanisms involved in thermostability of enzymes can be obtained (22,31,37).We selected the neutral proteases from bacilli as a model system in such an approach. Neutral proteases (Npr) are metalloendoproteinases which show optimum activity at neutral pH. These enzymes contain one zinc atom per molecule and may be stabilized by calcium binding (17). Several npr genes have been cloned and expressed in Bacillus subtilis, including the genes from B. subtilis, Bacillus stearothermophilus and Bacillus amyloliquefaciens, and their nucleotide sequences have been determined (8,12,18,33,38,40,44). Recently, the npr gene from Bacillus brevis was also cloned and sequenced (la). Additionally, the primary and tertiary structures of the neutral proteases from Bacillus cereus and Bacillus thermoproteolyticus have been determined (21,28,34,35). The neutral proteases from B. subtilis and B. amyloliquefaciens are rather thermolabile, whereas the enzymes from B. stearothermophilus and ...

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Section: Discussionmentioning

confidence: 99%

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product

Burg¹,

Enequist²,

Haar³

et al. 1991

J Bacteriol

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“…Molecular Biology-The nprT gene encoding the TLP of B. stearothermophilus CU21 (25) (TLP-ste) was cloned, subcloned, and expressed as described previously (26). Site-directed mutagenesis was performed on subcloned fragments of the TLP-ste gene using the QuikChange site-directed mutagenesis kit from Stratagene, La Jolla, CA.…”

Section: Modeling and Mutantmentioning

confidence: 99%

“…Site-directed mutagenesis was performed on subcloned fragments of the TLP-ste gene using the QuikChange site-directed mutagenesis kit from Stratagene, La Jolla, CA. The nucleotide sequences of mutated fragments of the nprT gene were verified by DNA sequencing and the mutated fragments were subsequently cloned into variants of the Bacillus expression vector pGE501 (26) containing the TLP-ste gene with a deletion of the previously subcloned fragment.…”

Section: Modeling and Mutantmentioning

confidence: 99%

The Effects of Modifying the Surface Charge on the Catalytic Activity of a Thermolysin-like Protease

Kreij

Burg

Venema

et al. 2002

Journal of Biological Chemistry

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The impact of long range electrostatic interactions on catalysis in the thermolysin-like protease from Bacillus stearothermophilus was studied by analyzing the effects of inserting or removing charges on the protein surface. Various mutations were introduced at six different positions, and double-mutant cycle analysis was used to study the extent to which mutational effects were interdependent. The effects of single point mutations on the k cat /K m were non-additive, even in cases where the point mutations were located 10 Å or more from the active site Zn 2؉ and separated from each other by up to 25 Å. This shows that catalysis is affected by large electrostatic networks that involve major parts of the enzyme. The interdependence of mutations at positions as much as 25 Å apart in space also indicates that other effects, such as active site dynamics, play an important role in determining active site electrostatics. Several mutations yielded a significant increase in the activity, the most active (quadruple) mutant being almost four times as active as the wild type. In some cases the shape of the pH-activity profile was changed significantly. Remarkably, large changes in the pH-optimum were not observed.The acceleration of reaction rates by enzymes is one of the essential prerequisites for life as we know it, and the multitude and diversity of enzymes shows that it should be possible to design an enzyme that will catalyze almost any reaction under almost any set of conditions. To achieve a high rate of acceleration, enzymes rely on charged groups in their active site that stabilize the transition state or function as acid or base catalysts in the reaction. The kinetic parameters of enzymes therefore display a significant pH dependence, which is determined by the pK a values of the active site groups.Catalysis depends on intricate electrostatic interactions, which may be noticeable over distances that are large compared with short range of interactions such as hydrogen bonds and hydrophobic contacts. Thus, larger parts of an enzyme may be involved in optimizing its catalytic center than previously thought. The long range character of electrostatic effects is illustrated by a, very limited, number of examples in the literature, showing that changes in surface charge at locations as far as 15 Å from a catalytic center may affect enzyme activity (1, 2). Unfortunately, electrostatic interactions are hard to handle theoretically not only because of their long range character but also because of intrinsic theoretical difficulties. For example, most electrostatic models still use a single rigid protein structure and, at most, two dielectric constants to account for all the dynamics of the protein. This clearly is an oversimplification of reality (3).We have studied the contribution of long range electrostatic interactions to catalysis by analyzing the effects of a series of charge mutations scattered over a larger part of the surface of a thermolysin-like protease from Bacillus stearothermophilus (TLP-ste).

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“…The protease-deficient strain Bacillus subtilis DB117 43 was used as host for this plasmid, and its variants were obtained by site-directed mutagenesis. Cells harboring these plasmids were grown at 37°C in TY broth containing 5 g/ml chloramphenicol as antibiotic.…”

Section: Reagents-14-dithio-dl-threitol (Dtt) and N-(3-[2-furyl]acrymentioning

confidence: 99%

“…Expression and Purification of Wild Type and Mutant EnzymesWild type and mutant proteins were produced and purified as described previously (43,48) using affinity chromatography on Bacitracin-silica. The enzymes were stored in elution buffer containing 20 mM sodium acetate, pH 5.3, 5 mM CaCl 2 , 2.5 M NaCl, 20% (v/v) isopropanol, and 0.03% (w/v) sodium azide.…”

Section: Reagents-14-dithio-dl-threitol (Dtt) and N-(3-[2-furyl]acrymentioning

confidence: 99%

Extreme Stabilization of a Thermolysin-like Protease by an Engineered Disulfide Bond

Mansfeld

Vriend

Dijkstra

et al. 1997

Journal of Biological Chemistry

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174

140

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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.

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Contribution of the C-terminal amino acid to the stability of Bacillus subtilis neutral protease

Cited by 39 publications

References 20 publications

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product

A highly thermostable neutral protease from Bacillus caldolyticus: cloning and expression of the gene in Bacillus subtilis and characterization of the gene product

The Effects of Modifying the Surface Charge on the Catalytic Activity of a Thermolysin-like Protease

Extreme Stabilization of a Thermolysin-like Protease by an Engineered Disulfide Bond

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