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PubMed2000

Novel oxidatively stable subtilisin-like serine proteases from alkaliphilic Bacillus spp.: enzymatic properties, sequences, and evolutionary relationships.

The genes for five subtilisin-like serine proteases from alkaliphilic strains of Bacillus exhibiting resistance to oxidative inactivation were cloned and sequenced. The deduced amino acid sequences of the enzymes were highly homologous (greater than 88% identity). They were composed of 638 or 639 amino acids, including a possible approximately 200-amino acid prepro-peptide, and unique stretches of approximately 160 amino acids were found in the C-terminal regions. The molecular masses of mature enzymes (433 or 434 amino acids) were approximately 45 kDa for all. Amino acid sequence comparison and phylogenetic analysis indicated that these enzymes are far removed from other known subtilisins in the line of molecular evolution. We propose that these novel proteases be categorized as a new class of subtilisins, named oxidatively stable, alkaline protease.

Amino Acid Sequence↗
PubMed1994

Cavity mutants of Savinase. Crystal structures and differential scanning calorimetry experiments give hints of the function of the buried water molecules in subtilisins.

The subtilisin molecule possesses several internal water molecules, which may be characterised as an integral part of the protein structure. We have introduced specific mutations (T71I, T71S, T71V, T71A and T71G) at position 71 in the subtilisin variant Savinase from Bacillus lentus. This position is involved in a hydrogen bonded network with several internal water molecules, forming a water channel. The water channel and most of the other internal water molecules are positioned in the interface between two half-domains of the subtilisin molecule. The data presented here indicate that the internal water molecules are structural, and may be the result of trapping during the folding process.

Amino Acids↗
PubMed1999

Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacillus Ak.1 protease at 1.8 A resolution.

Proteins of the subtilisin superfamily (subtilases) are widely distributed through many living species, where they perform a variety of processing functions. They are also used extensively in industry. In many of these enzymes, bound calcium ions play a key role in protecting against autolysis and thermal denaturation. We have determined the crystal structure of a highly thermostable protease from Bacillus sp. Ak.1 that is strongly stabilized by calcium. The crystal structure, determined at 1.8 A resolution (R=0. 182, Rfree=0.247), reveals the presence of four bound cations, three Ca(2+) and one Na(+). Two of the Ca(2+) binding sites, Ca-1 and Ca-2, correspond to sites also found in thermitase and the mesophilic subtilisins. The third calcium ion, however, is at a novel site that is created by two key amino acid substitutions near Ca-1, and has not been observed in any other subtilase. This site, acting cooperatively with Ca-1, appears to give substantially enhanced thermostability, compared with thermitase. Comparisons with the mesophilic subtilisins also point to the importance of aromatic clusters, reduced hydrophobic surface and constrained N and C termini in enhancing the thermostability of thermitase and Ak.1 protease. The Ak.1 protease also contains an unusual Cys-X-Cys disulfide bridge that modifies the active site cleft geometry.

Amino Acid Sequence↗
PubMed2002

Structure of POIA1, a homologous protein to the propeptide of subtilisin: implication for protein foldability and the function as an intramolecular chaperone.

Solution structure of POIA1 (Pleurotus ostreatus proteinase A inhibitor 1), which functions as an intramolecular chaperone and as an inhibitor to subtilisin, was determined. By making use of the fact that POIA1 is the only structured protein that shows homology to the propeptide of subtilisin, which is unstructured by itself, foldability of this protein was elucidated. It became clear that the evolutionarily conserved residues play two important roles, one for the maintenance of its own structure, and the other for the interaction with subtilisin. Structural softness and mutational tolerance contained in the POIA1 structure makes it an ideal material for designing a foldable protein.

Amino Acid Sequence↗
PubMed1993

Basic trypsin-subtilisin inhibitor from marine turtle egg white: hydrodynamic and inhibitory properties.

A basic trypsin-subtilisin inhibitor has been isolated from the egg white of marine turtle (Caretta caretta Linn.) and purified to homogeneity by gel filtration followed by ion-exchange chromatography. It has a single polypeptide chain of 117 amino acid residues, having a molecular weight of 13,600. It lacks methionine and tryptophan. Its isoelectric point is at pH 10.0 and the sedimentation coefficient (S20,w) value of 1.62 S is independent of protein concentration. It has a Stokes radius of 18.8 A, an intrinsic viscosity of 0.048 dl g-l and a diffusion coefficient of 10.17 x 10(-7) cm2 sec-1. Its fluorescence emission spectrum is similar to that of free tyrosine and the bimolecular quencing rate constant of its tyrosine residues with acrylamide is 3.15 x 10(9) M-1 sec-1. The inhibitor strongly inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio of unity. The nature of inhibition toward both enzymes is not temporary. It has independent binding sites for inhibition of trypsin and subtilisin. Chemical modification with tetranitromethane suggests the presence of three tyrosine residues on the surface of the inhibitor molecule.

Amino Acids↗
PubMed1996

A serine alkaline protease from the fungus Conidiobolus coronatus with a distinctly different structure than the serine protease subtilisin Carlsberg.

In view of the functional similarities between subtilisin Carlsberg and the alkaline protease from Conidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, the Conidiobolus protease is structurally distinct from subtilisin Carlsberg. The Conidiobolus protease was also different from other bacterial and animal proteases (e.g. pronase, protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus protease antibody in double diffusion and in neutralization assays. The Conidiobolus serine protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal proteases.

Amino Acids↗
PubMed1997

Cold adaptation of a mesophilic serine protease, subtilisin, by in vitro random mutagenesis.

Artificial cold adaptation of a mesophilic protease, subtilisin BPN' was attempted by means of random mutagenesis of its entire gene coupled with screening of cleared-zone-forming colonies on skim-milk plates at a low temperature. Out of sixty clones screened at 10 degrees C, one mutant enzyme (termed M-15) was found to acquire higher proteolytic activities, specifically dependent on low temperatures ranging from 10 degrees C to 1 degree C, in comparison with those of the wild-type. DNA sequencing analysis revealed that, by this mutation, the 84th amino acid residue, valine, was substituted by isoleucine, which is located 1.5 nm from the center of the catalytic triad in the tertiary structure of subtilisin. By kinetic analysis of the purified enzyme samples, the higher proteolytic activities of M-15 at low temperatures were found to be due to the decrease in the Km value. There was no difference in thermostability between the wild-type and mutant enzymes, when tested by heat treatment. Circular dichroism spectra also showed no difference between them at 10 degrees C, indicating that the mutation of V841 had no effect on the secondary structure of subtilisin.

Adaptation, Biological↗
PubMed2000

The seed coat-specific expression of a subtilisin-like gene, SCS1, from soybean.

A seed coat-specific gene, SCS1 (Seed Coat Subtilisin 1), from soybean, Glycine max [L.] Merill, has been identified and studied. The gene belongs to a small family of genes with sequence similarity to the subtilisins, which are serine proteases. Northern blot analysis showed that SCS1 RNA accumulates to maximal levels in seed coats at 12 days post anthesis, preceding the final stages of seed coat differentiation. The SCS1 RNA was not found in other tissues including embryos, seed pods, flowers, stems, roots or leaves. In-situ hybridization studies confirmed the temporal pattern of expression observed by Northern blot analysis and further revealed a restricted pattern of RNA accumulation in thick-walled parenchyma cells of the seed coats. These cells are important in the apoplastic translocation of nutrients en route to the embryo from the vascular tissues. The tissue-specific subtilisin-like gene may be required for regulating the differentiation of the thick-walled parenchyma cells.

Amino Acid Sequence↗
PubMed2004

A new subtilisin family: nucleotide and deduced amino acid sequences of new high-molecular-mass alkaline proteases from Bacillus spp.

Six genes encoding high-molecular-mass subtilisins (HMSs) of alkaliphilic Bacillus spp. were cloned and sequenced. Their open reading frames of 2,394-2,424 bp encoded prosubtilisins of 798-808 amino acids (aa) consisting of the prepropeptides of 151-158 aa and the mature enzymes of 640-656 aa. The deduced aa sequences of the mature enzymes exhibited 60-95% identity to those of FT protease of Bacillus sp. strain KSM-KP43, a subtilisin-like serine protease, and a minor serine protease, Vpr, of Bacillus strains. Three of the six recombinant enzymes were susceptible to proteolysis, but the others were autodigestion resistant. All enzymes had optimal pH values of 10.5-11.0, optimal temperatures of 40-45 degrees C for hydrolysis of a synthetic substrate, and were heat labile. These alkaline proteases seem to form a new subtilisin family, as judged by their aa sequences and phylogenetic analysis.

Amino Acid Sequence↗
PubMed1985

Purification of subtilisin by single-step affinity chromatography.

The dye 4-(4-aminophenylazo)phenylarsonic acid was coupled to activated CH-Sepharose 4B and the resulting affinity matrix was shown to be highly efficient for the purification of subtilisin. A crystalline subtilisin was purified to homogeneity using this affinity chromatography procedure with a purification fold of 1.4 and with an enzyme activity yield of 98%. Similarly subtilisin from a crude enzyme preparation was purified to 211 fold by this single step procedure with 94% recovery of the enzyme activity. The purified enzymes were shown to be homogeneous by polyacrylamide gel electrophoresis.

Arsenicals↗
PubMed1978

Binding of m-nitrobenzeneboronic acid to the active site of subtilisin BPN.

m-Nitrobenzeneboronic acid as a possible transition-state analog for serine proteases was found to cause absorption spectral change from 250 nm 350 nm upon binding with subtilisin BPN' (EC 3.4.21.14) at pH 6.5. Similar difference spectral changes of m-nitrobenzeneboronic acid were also observed at alkaline pH or upon addition of N-methylimidazole at pH 6.5. A characteristic circular dichroism spectrum of m-nitrobenezeneboronic acid was induced upon binding with subtilisin BPN' not only at pH 6.5, but also at alkaline pH. Circular dichroism spectral titration confirmed the stoichiometry of 1 : 1 for the m-nitrobenzeneboronic acid - subtilisin complex. m-Nitrobenzeneboronic acid was shown to be useful as a reversible chromophoric probe for the catalytic site of serine proteases.

Binding Sites↗
PubMed1980

Independent heat stabilization of proteases associated with multiheaded inhibitors. Complexes of chymotrypsin, subtilisin and trypsin with chicken ovoinhibitor and with lima bean protease inhibitor.

The heat stabilization resulting from specific association of serine proteases with either of two multiheaded protease inhibitors, chicken ovoinhibitor or lima bean protease inhibitor, was determined at pH 6.7 in a differential scanning calorimeter. The 2:1 complex of either bovine alpha-chymotrypsin or subtilisin BPN' with ovoinhibitor showed two major denaturation endotherms; each 1:1 complex showed one major endotherm. Association with ovoinhibitor increased the kinetic thermal stabilities over those of the free chymotrypsin or subtilisin. Association with lima bean protease inhibitor stabilized bovine beta-trypsin greater than porcine beta-trypsin greater than bovine alpha-chymotrypsin. Complexes having different proteases bound to the same inhibitor, such as chymotrypsin . ovoinhibitor . subtilisin (1:1:1) or trypsin . inhibitor . chymotrypsin (1:1:1), denatured like mixtures of the 1:1 complexes. These results show more clearly that 2:1 association with multiheaded inhibitors stabilizes the two bound protease molecules independently. Each bound protease and the domain(s) of the inhibitor influenced by specific binding of this protease are denatured as a unit. Thus, 2:1 complexes comprise at least two new denaturing units. The extent of heat stabilization appears roughly proportional to the Kassoc determined by other methods. The results are consistent with other evidence that binding sites for proteases on multi-headed inhibitors are relatively independent in structure and function.

Animals↗
PubMed1976

Specific fluorescent derivatives of macromolecules. A fluorescence study of some specifically modified derivatives of chymotrypsin, trypsin and subtilisin.

The 5-dimethylaminonaphthalene-1-sulfonyl group was specifically introduced into the active site region of the serine proteinases: alpha-chymotrypsin, trypsin and subtilisin Carlsberg by the method of affinity-labeling. The resulting fluorescent derivatives were studied by a variety of fluorescence techniques and the results were correlated with structural data available on these enzymes from X-ray analysis. As model compounds for the Dns-proteinases, the absorption and fluorescence properties of Dns-amide and Dns-ethyl ester were studied in ethanol/water and p-dioxane/water mixtures. The fluorescence emission transtion energies and quantum yields were related to four commonly employed solvent-polarity scales. Best correlations for different solvents were obatined with the empirical "Z" and "Y" scales. From inspection of the fluorescence emission transition energies of the Dns group in the Dns-proteinases and comparision with the model compound studies it was possible to assign "Z" values for the apparent microenvironment polarities of the Dns group in the Dns-proteinases. The apparent polarities of the microenvironments of the Dns group in Dns-Ser 195-chymotrypsin (Dns-chymotrypsin (I)); (Dns-Phe-CH2)-His 57-chymotrypsin; (Dns-Lys-CH2)-His 46-trypsin; and Dns-Ser 221--subtilisin Carlsberg (Dns-subtilisin (I)) are in the range of 89.5-92.5 on the "Z" scale. The apparent microenvironment polarity of the Dns group in Dns-Ser 183-trypsin (Dns-trypsin (I)) appears to be below 76.7 on the "Z" scale. The Dns group in Dns-chymotrypsin (I) and (Dns-Phe-CH2)-His 57-chymotrypsin appears to be rigidly bound as evaluated by fluorescence polarization studies. The effect of 2H2O on the fluorescence emission quantum yields of Dns-amide and Dns-ethyl ester was examined. In both cases the ratios of quantum yields in 2H2O:ethanol (8:2) to quantum yields in H2O:ethanol (8:2) was about 1.8. The 2H2O effect upon the fluorescence emission quantum yields of the Dns group has been used to investigate solvent accessibility of this chromophore in the Dns-proteinases. Acessibility studies using 2H2O are very promising and have some definite advantages over other existing methods. Energy transfer between the Trp residues and the bound Dns group was investigated in the Dns-proteinases. The mean transfer distance calculated from the observed transfer efficiencies are 18.1 A, 19.7 A and 18.4 A for (Dns-Phe-CH2)-His 57-chymotrypsin, Dns-chymotrypsin (I) and (Dns-Lys-CH2)-His 46-trypsin, respectivly. From models built using X-ray crystallographic coordinates for the protein atoms, the mean distance of separation between the Trp residues and the bound Dns group for the same set of conjugates ar 18.6 A, 17.5 A and 17.5 A, respectively. Considering the inherent difficulties in energy transfer studies, the results are in excellent agreement with the X-ray data.

Chymotrypsin↗
PubMed1992

Effect of the weak Ca(2+)-binding site of subtilisin J by site-directed mutagenesis on heat stability.

The functional role of the negatively charged amino acid residue in subtilisin J from Bacillus stearothermophilus has been investigated by site-directed mutagenesis. Glu-195 located at the weak Ca2+-binding site was replaced with Gln to examine the role of Glu-195 in the heat stability of subtilisin J. Mutant enzyme was expressed in Bacillus subtilis and was purified from the culture supernatant. When the mutant enzyme was expressed at 37 degrees C in the presence of 2mM calcium chloride, the pattern of enzyme production was quite different from that of wild-type. The purified Gln-195 mutant enzyme was analyzed with respect to optimal temperature, optimal pH, and heat stability. The mutation was found to decrease the heat stability but not catalytic efficiency (kcat/Km) and optimal pH. These results demonstrate the important role of the negatively charged side chains at the weak Ca(2+)-binding site in the heat stability of subtilisin.

Amino Acid Sequence↗
PubMed1988

Circular dichroism and fluorescence of polyethylene glycol-subtilisin in organic solvents.

Subtilisin Carlsberg has been made soluble in organic solvents such as dioxane and acetonitrile by covalent linking to polyethylene glycol. Far-ultraviolet circular dichroism and intrinsic protein fluorescence have shown that subtilisin dissolved in dioxane, in which the enzyme is active and highly stable, maintains the native secondary structure as well as the native microenvironment for tyrosyl residues. In acetonitrile subtilisin undergoes conformational changes that cause enzyme inactivation and precipitation.

Acetonitriles↗
PubMed1992

Structure of tubulin C-terminal domain obtained by subtilisin treatment. The major alpha and beta tubulin isotypes from pig brain are glutamylated.

Limited subtilisin digestion of the tubulin alpha, beta heterodimer has been used in this work to reduce the total number of tubulin isotypes from 20 for native to 9 for subtilisin-cleaved tubulin. This indicates that the major part of tubulin heterogeneity is located at the C-terminus of the molecule. The C-terminal peptides of both alpha and beta subunits of tubulin were purified by anion-exchange HPLC. Combined use of Edman degradation chemistry and mass spectrometry on the isolated peptides shows that subtilisin cleavage occurs at position Asp-438 and His-406 of alpha and Gln-433 and His-396 of beta tubulin chains. Quantitative analysis of our data show that cleavage at positions His-406 (alpha) and His-396 (beta) occurs with a low efficiency and indicates that the major isotypes of pig brain tubulin are modified by sequential attachment of 1 to 5 glutamic acid residues at positions Glu-445 or -435 of alpha and beta tubulin, respectively.

Amino Acid Sequence↗
PubMed1993

The antigenic domain of flagellin from S. paratyphi shares a structural fold with subtilisin.

Bacterial flagellin has two domains: the polymerizing domain consisting of N- and C-terminal regions which are partly disordered in the monomeric state; and the central antigenic domain with compact globular structure. The polymerizing domain is highly conserved in flagellins from different species but the antigenic domain is diverse in sequence and size. Whereas the former has direct functional significance for bacterial motility, the latter has not been identified as having a specific function except for defining the distinct serotype of the bacterium. The sequence alignment of flagellin from S. paratyphi with proteins of known three-dimensional structure reveals significant homology of the central 265 residue stretch with the bacterial serine protease, subtilisin. This homology is evident also in the comparison of the predicted secondary structure of flagellin with the observed secondary structural features in subtilisin. The deletions/insertions arising due to optimal alignment of the two proteins occur on the surface loops in the structure. Thus, a domain of S. paratyphi flagellin and subtilisin appear to have similar structural folds.

Amino Acid Sequence↗
PubMed1994

Contribution of salt bridge in the protease inhibitor SSI (Streptomyces subtilisin inhibitor) to its inhibitory action.

The tertiary structure of proteinaceous protease inhibitors is considered to be maintained by various interactions in the molecule that prevent degradation by protease. In this study, the Arg29 of Streptomyces subtilisin inhibitor (SSI) forming a salt bridge with the carboxyl group of carboxyl-terminal Phe113 was replaced with Ala, Met or Lys by cassette mutagenesis to clarify the role of Arg29 in the function of SSI. The inhibitory activity of each mutated SSI decreased with increasing incubation time after mixing with subtilisin, indicating that the SSI was changed into a temporary inhibitor upon mutation. This decrease was shown by SDS polyacrylamide gel electrophoresis to be due to cooperative degradation of the mutated SSI by subtilisin. In addition, the denaturation temperature of the Ala or Met mutant was decreased by ten degrees and that of the Lys mutant by 1.5 degrees, suggesting that the destabilization of SSI may be related to its temporary inhibition. Thus, interaction in the protease inhibitor molecule for maintaining the tertiary structure, such as that of Arg29 in SSI, was shown to be required for the inhibitory action.

Alanine↗