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.     

Effects of changing the interaction between subdomains on the thermostability of Bacillus neutral proteases.

Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to alter the interaction between the middle and C-terminal subdomain of these enzymes. In all Nprs a cluster of hydrophobic contacts centered around residue 315 contributes to this interaction. In thermostable Nprs (like Npr-ste) a 10 residue beta-hairpin, covering the domain interface, makes an additional contribution. The hydrophobic residue at position 315 was replaced by smaller amino acids. In addition, the beta-hairpin was deleted from Npr-ste and inserted into Npr-sub. The changes in thermostability observed after these mutations confirmed the importance of the hydrophobic cluster and of the beta-hairpin for the structural integrity of Nprs. Combined mutants showed that the effects of individual mutations affecting the interaction between the subdomains were not additive. The effects on thermostability decreased as the strength of the subdomain interaction increased. The results show that once the subdomain interface is sufficiently stabilized, additional stabilizing mutations at the same interface do not further increase thermostability. The results are interpreted on the basis of a model for the thermal inactivation of neutral proteases, in which it is assumed that inactivation results from the occurrence of local unfolding processes that render these enzymes susceptible to autolysis.     

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

The role of the C-terminal Leu300 in maintaining thermal stability of the neutral protease of Bacillus subtilis was investigated. From model building studies based on the three-dimensional structure of thermolysin, the neutral protease of B. thermoproteolyticus, it was concluded that this residue is located in a hydrophobic pocket composed of residues located in the C-terminal and the middle domain. To test the hypothesis that Leu300, by contributing to a stabilizing interaction between these domains, is important for enzyme stability, several neutral protease mutants were constructed and characterized. The thermostability of the enzyme was lowered by deleting Leu300 or by replacing this residue by a smaller (Ala), a polar (Asn) or a sterically unfavourable (Ile) amino acid. Thermostability was increased upon replacing Leu300 by Phe. These results are in agreement with model-building studies. The effects on thermostability observed after mutating the corresponding Val318 in the thermostable neutral protease of B.stearothermophilus were less pronounced.     

The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase

The complete amino-acid sequence of a neutral proteinase, produced by Bacillus cereus, was determined by protein sequencing. The neutral proteinase consists of 317 amino-acid residues. The primary structure is 70% homologous to thermolysin, a thermostable neutral proteinase and 45% homologous to Bacillus subtilis neutral proteinase. The zinc-binding site and the hydrophobic pocket of the active site are highly similar in all three proteinases. B. cereus neutral proteinase which is 20 degrees C less thermostable (60 degrees C) than thermolysin (80 degrees C) shows only minor differences in calcium binding sites and salt bridges compared to thermolysin (known from its X-ray diffraction analysis), whereas B. subtilis neutral proteinase (50 degrees C) differs considerably. Therefore it was assumed that the difference in thermostability between B. cereus neutral proteinase and thermolysin is not caused by different metal binding properties, or differences in the active site, but by changes within the rest of the molecule. Calculation of secondary structure potentials according to Chou & Fasman, hydrophobicity and bulkiness of the different structural elements and preferred cold----hot amino-acid residue exchanges indicated, that the thermostability of thermolysin compared to B. cereus neutral proteinase is caused by small effects contributed by numerous amino-acid exchanges distributed over the whole molecule, resulting in increased hydrophobicity of beta-pleated sheet and higher bulkiness of alpha-helical regions.     

The structure of neutral protease from Bacillus cereus at 0.2-nm resolution.

The crystal structure of the neutral protease from Bacillus cereus has been refined to an R factor of 17.5% at 0.2-nm resolution. The enzyme, an extracellular metalloendopeptidase, consists of two domains and binds one zinc and four calcium ions. The structure is very similar to that of thermolysin, with which the enzyme shares 73% amino-acid sequence identity. The active-site cleft between the two domains is wider in neutral protease than in thermolysin. This suggests the presence of a flexible hinge region between the two domains, which may assist enzyme action. The high-resolution analysis allows detailed examination of possible causes for the difference in thermostability between neutral protease and thermolysin.     

Crystal structure of neutral protease from Bacillus cereus refined at 3.0 A resolution and comparison with the homologous but more thermostable enzyme thermolysin

Neutral protease from Bacillus cereus exhibits a 73% amino acid sequence homology to thermolysin, for which an accurate crystal structure exists. The B. cereus enzyme is, however, markedly less thermostable. The neutral protease was crystallized and diffraction data to 3.0 A resolution were recorded by oscillation photography. The crystal structure was solved by molecular replacement methods using thermolysin as a trial molecule. The solution was improved by rigid-body refinement and model rebuilding into electron density omit-maps. The atomic co-ordinates were refined to R = 21.7% at 3.0 A resolution. Comparison of the resultant model with the thermolysin structure shows that the two enzymes are very similar with a root-mean-square deviation between equivalent C alpha-atoms of 0.88 A. The gamma-turn found in thermolysin is transformed into a beta-turn in the neutral protease by the insertion of a glycine residue. There appear to be no contributions to the enhanced thermostability of thermolysin from additional salt bridges, whereas contributions in the form of extra hydrogen bonding interactions could be important. Other factors that may affect thermostability include the two glycine to alanine exchanges and perturbations in the environment of the double calcium site.     

Thermostability of Bacillus subtilis neutral protease.

The thermostability of the B. subtilis neutral protease was studied under various conditions. At elevated temperatures the enzyme was inactivated as a result of autolysis. The rate of inactivation did not depend on the enzyme concentration and the enzyme was most stable near its pH optimum. The rate of inactivation was unaffected by the presence of a second protease during the incubation at high temperatures. The results indicate that the rate of thermal inactivation of the neutral protease is determined by the kinetics of local unfolding processes that precede autolysis rather than by the catalytic rate of the autodigestion reaction or an irreversible unfolding step.     

A simple electrostatic criterion for predicting the thermal stability of proteins

The enhancement of protein thermostability is an important issue for both basic science and biotechnology purposes. We have developed a thermostability criterion for a protein in terms of a quasi-electric dipole moment (contributed by its charged residues) defined for an electric charge distribution whose total charge is not zero. It was found that minimization of the modulus of this dipole moment increased its thermal stability, as demonstrated by surveying these values in pairs of mesostable-thermostable homologous proteins and in mutations described in the literature. The analysis of these observations provides criteria for thermostabilization of a protein, by computing its dipole profile. This profile is obtained by direct substitution of each amino acid of the sequence by either a positive, negative or neutral amino acid, followed by a recalculation of the dipole moment. As an experimental example, these criteria were applied to a beta-glucanase to enhance its thermal stability.     

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

By using a gene library of Bacillus caldolyticus constructed in phage lambda EMBL12 and selecting for proteolytically 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 subtilis strain resulted in protease proficiency of the host. The nucleotide sequence of a 2-kb HinfI-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. sterothermophilus 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 degrees 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 thermostability 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.     

Perturbation of conformational dynamics,enzymatic activity,and thermostability of β-glycosidase from archaeon Sulfolobus solfataricus by pH and sodium dodecyl sulfate detergent

The conformational dynamics of β-glycosidase from Sulfolobus solfataricus was investigated by following the emission decay arising from the large number of tryptophanyl residues that are homogeneously dispersed in the primary structure. The fluorescence emission is characterized by a bimodal lifetime distribution, suggesting that the enzyme structure contains rigid and flexible regions, properly located in the macromolecule. The enzyme activity and thermostability appear to be related to the dynamic properties of these regions as evidenced by perturbation studies of the enzyme structure at alkaline pH and by addition of detergents such as SDS. The pH increase affects the protein dynamics with a remarkable loss of thermal stability and activity; these changes occur without any significant variation in the secondary structure as revealed by far-UV dichroic measurements. In the presence of 0.02% (w/v) SDS at alkaline pH, the enzymatic activity and thermostability are recovered. Under these conditions, the conformational dynamics appear to be similar to that evidenced at neutral pH. Further increases in SDS concentration, at alkaline pH, render the activity and thermostability of β-glycosidase similar to those observed in the absence of detergent. Proteins 27:71–79 © 1997 Wiley-Liss, Inc.     

Stabilization of the neutral protease of Bacillus stearothermophilus by removal of a buried water molecule.

Using site-directed mutagenesis, Ala166 in the neutral protease of Bacillus stearothermophilus was changed into Ser. Model building and molecular dynamics simulations of the mutant enzyme indicated that the Ser hydroxyl group fits well in a cavity which contains a water molecule in the wild-type enzyme. The Ala166----Ser mutation was expected to exert a stabilizing effect because of the gain in entropy resulting from the release of water molecule from the folded protein to the solvent. In addition, the hydrogen-bonding network around residue 166 was improved upon the mutation. As a result of this mutation the thermostability of the neutral protease was increased by 1.2 +/- 0.1 degrees C.     

Influence of protein conformation on its adaptability under chaotropic conditions

The thermostability of serum albumin and beta-lactoglobulin in various salt solutions was studied using differential scanning calorimetry. Below 1.0 M salt concentrations, the relative effectiveness of various sodium salts on increasing the thermostability of beta-lactoglobulin followed the classic Hofmeister or lyotropic series, i.e. SO2-(4) greater than Cl- greater than Br- greater than ClO-4 greater than SCN-; however, in the case of serum albumin the above order was reversed, i.e. ClO-4 greater than SCN- greater than Br- greater than Cl- greater than SO2-(4), indicating that the thermostability of serum albumin was higher in chaotropic solution conditions. Circular dichroic analysis of serum albumin in NaClO4 solutions revealed that the alpha-helical content of the protein increased from 59% to 73% in 1.0 M NaClO4; no similar increase in secondary structure was observed for beta-lactoglobulin. These observations contradicted the general notion that the chaotropic effect of neutral salts on the stability of macromolecules is independent of any details of the macromolecular conformation itself. The results presented here indicate that the predisposition of the native conformation of a protein per se might affect whether the protein would undergo stabilization or destabilization (i.e. conformational adaptability) under moderate chaotropic solution conditions.     

In silico characterization of thermostable lipases

Thermostable lipases are of high priority for industrial applications as they are endowed with the capability of carrying out diversified reactions at elevated temperatures. Extremophiles are their potential source. Sequence and structure annotation of thermostable lipases can elucidate evolution of lipases from their mesophilic counterparts with enhanced thermostability hence better industrial potential. Sequence analysis highlighted the conserved residues in bacterial and fungal thermostable lipases. Higher frequency of AXXXA motif and poly Ala residues in lid domain of thermostable Bacillus lipases were distinguishing characteristics. Comparison of amino acid composition among thermostable and mesostable lipases brought into light the role of neutral, charged and aromatic amino acid residues in enhancement of thermostability. Structural annotation of thermostable lipases with that of mesostable lipases revealed some striking features which are increment of gamma turns in thermostable lipases; being first time reported in our paper, longer beta strands, lesser beta-branched residues in helices, increase in charged-neutral hydrogen bonding pair, hydrophobic-hydrophobic contact and differences in the N-cap and C-cap residues of the α helices. Conclusively, it can be stated that subtle changes in the arrangement of amino acid residues in the tertiary structure of lipases contributes to enhanced thermostability.     

Molecular strategies for protein stabilization: the case of a trehalose/maltose-binding protein from Thermus thermophilus

The trehalose/maltose-binding protein (MalE1) is one component of trehalose and maltose uptake system in the thermophilic organism Thermus thermophilus. MalE1 is a monomeric 48 kDa protein predominantly organized in alpha-helix conformation with a minor content of beta-structure. In this work, we used Fourier-infrared spectroscopy and in silico methodologies for investigating the structural stability properties of MalE1. The protein was studied in the absence and in the presence of maltose as well as in the absence and in the presence of SDS at different p(2)H values (neutral p(2)H and at p(2)H 9.8). In the absence of SDS, the results pointed out a high thermostability of the MalE1 alpha-helices, maintained also at basic p(2)H values. However, the obtained data also showed that at high temperatures the MalE1 beta-sheets underwent to structural rearrangements that were totally reversible when the temperature was lowered. At room temperature, the addition of SDS to the protein solution slightly modified the MalE1 secondary structure content by decreasing the protein thermostability. The infrared data, corroborated by molecular dynamics simulation experiments performed on the structure of MalE1, indicated that the protein hydrophobic interactions have an important role in the MalE1 high thermostability. Finally, the results obtained on MalE1 are also discussed in comparison with the data on similar thermostable proteins already studied in our laboratories.     

Isolation and characterization of a bacteriocin produced by Bacillus stearothermophilus strain NU-10.

Broth-grown cultures of Bacillus stearothermophilus strain NU-10 produce a bacteriocin which exerts lethal activity on other strains of the bacterium. Optimal production occurs during late maximum stationary phase of growth, at neutral pH, and 55-65 degrees C. The bacteriocin can be substantially purified by a combination of precipitations, centrifugations, and gel filtrations. The thermocin is composed of protein and carbohydrate. It is partially destroyed by proteolytic enzymes but is resistant to DNase, RNase, and various chemical treatments. The bacteriocin has a small molecular weight and exhibits considerable thermostability.     

Characterisation of a thermostable alpha-amylase from Bacillus brevis.

Biochemical characterization of a novel heat-stable alpha-amylase, produced by a thermophilic strain of Bacillus brevis, has been made. The pattern of the enzyme action on different substrates was studied. It was found that reducing groups were rapidly liberated from amylopectin, soluble and insoluble starch compared to amylose and glycogen. B. brevis alpha-amylase acted via endo-attack producing mainly maltopentaose during the first hour of hydrolysis. The enzyme showed high activity towards maltohexaose and maltoheptaose. The alpha-amylase from B. brevis had a neutral pI and was found to be a glycoprotein, containing 9.2% (by mass) neutral sugars. The enzyme protein possessed a unique high glycine content. Calcium or sodium ions in appropriate concentrations were required for enzyme thermostability.     

A new thermostable proteinase from Thermoactinomyces SP.27a. Cloning and gene expression

A library of Thermoactinomyces sp. 27a, producer of thermostable proteases of different groups, has been created. Gene coding for thermostable neutral proteinase was cloned and expressed in Bac. subtilis cells. Restriction map for cloned DNA fragment was created and physicochemical parameters of recombinant proteinase were characterized. The thermostability and optimum of proteolytic activity of the enzyme was lower than in the natural Thermoactinomyces sp. strain, which can be due to heterologous expression of the gene coding for thermostable protein in the mesophilic host.     

The fluctuation of various enzyme activities due to myo-inositol deficiency in Saccharomyces carlsbergensis.

The neutral lipid accumulation in myo-inositol deficient Saccharomyces carlsbergensis results at least partly from an enhancement of acetyl CoA carboxylase activity due to the high level of fructose 1,6-biphosphate which activates acetyl CoA carboxylase, and due to the low level of citrate which counteracts the activation [4]. In an attempt to explore the effect of myo-inositol deficiency on the metabolic fluxes, various enzyme activities were compared between the myo-inositol supplemented and deficient cells. The activities of phosphofructokinase and ATP-citrate lyase increased by 74 and 83%, respectively. The activity of glucose-6-phosphate dehydrogenase was unchanged. Unlike acetyl CoA carboxylase, elimination of low molecular effectors had no influence on their activities. The thermostability of phosphofructokinase (at 53 degrees C) increased, while that of aldolase (at 48 degrees C) greatly decreased due to the deficiency. The thermostability of glucose-6-phosphate dehydrogenase (at 52 degrees C) was also unchanged.     

Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues.

To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6- glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced. In spite of a big difference in their thermostabilities, B. coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an obligately thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%). The number of prolines (19 for B. cereus oligo-1,6-glucosidase, 24 for B. coagulans enzyme, and 32 for B. thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases. Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency. Judging from proline residues occurring in B. coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the beta turn and first turn of the alpha helix) (K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, and Y. Suzuki, Eur. J. Biochem. 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B. cereus oligo-1,6-glucosidase. With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory. The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues. It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases.