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PubMed1994

Purification and characterization of two Kunitz family subtilisin inhibitors from seeds of Canavalia lineata.

Two subtilisin inhibitors (CLSI-II and -III) were purified from seeds of Canavalia lineata by extraction with water, ammonium sulfate precipitation, and chromatographies on DEAE-Toyopearl and hydroxyapatite. The two inhibitors have the same molecular weight of about 22,000, and quite similar amino acid compositions. They contain five half-cystine residues and tend to dimerize through an intermolecular disulfide bridge due to the presence of a single cysteine residue. CLSI-III only inhibited subtilisin-type serine proteases, while CLSI-II showed a wider inhibitory specificity. Though the two inhibitors have almost identical thermal labilities, CLSI-II is more stable as to extreme pH than CLSI-III. They are considered to be Kunitz type inhibitors on the basis of several properties.

Amino Acid Sequence↗

PubMed1977

Crystal structure of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor, at 2.3 angstrom resolution.

The crystal structure of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor which strongly inhibits bacterial alkaline proteinases, was determined at 2.3 angstrom resolution. The subunit (molecular weight, 11,485) of this dimeric molecule has a unique fold of polypeptide chain with a five-fold anti-parallel beta-sheet structure (about 21% of the 113 amino acid residues) and two small segments of alpha-helices (about 16%). The region around the apparent reactive site, Met(73)-Val(74), is held tight by a combination of various structural features. The conformation of this region seems to have close similarity to that found in substrate analogues of low molecular weight bound to subtilisin BPN'.

Crystallography↗

PubMed1979

Interactions of BPN' and Carlsberg subtilisins with peptides containing aromatic amino acids at the C-terminus. Specific rate enhancement due to the secondary enzyme-substrate interaction.

The interaction between BPN' or Carlsberg subtilisins and peptides of the type Ac-Glyn-X-OMe (n = 0, 1, 2, 3), where X denotes one of five different aromatic amino acids, was investigated to elucidate the effect of the secondary interaction on catalysis in relation to the nature of the X residue. The increase in interaction upon elongation of the chain was accompanied by a large increase in kcat but with no marked change in Km in all the series of sensitive substrates. The peptides containing 2-(2-nitro-4-carboxyphenylsulfenyl)-tryptophan, however, acted as competitive inhibitors and exhibited an invariant dissociation constant in spite of the different chain lengths. These observations suggest that the secondary enzyme-substrate interaction induces a conformational change in the active site of the enzyme or in the substrate in such a way as to lower the activation energy and to form a stabilized transient complex. In this respect, BPN' and Carlsberg subtilisins are similar to porcine pepsin and Streptomyces griseus protease 1 rather than to alpha-chymotrypsin.

Calorimetry↗

PubMed1980

Importance of the carboxyl-terminal four amino acid residues in the inhibitory activity of Streptomyces subtilisin inhibitor (with a revision of its carboxyl-terminal sequence).

The carboxyl-terminal amino acid sequence of Streptomyces subtilisin inhibitor (SSI) was reinvestigated by analysis of the amino acid sequences of the thermolysin peptides from the C-terminal decapeptide, and the sequence -Val110-Ala-Phe-Phe113, which was reported in J. Biochem. 76, 1191-1209 (1974), was revised to -Val110-Phe-Ala-Phe113. Carboxypeptidase A digestion of SSI resulted in loss of the inhibitory activity in parallel with the release of the carboxyl-terminal four amino acid residues. The resulting modified inhibitor, des(Val110-Phe113)-SSI, possessed almost full inhibitory activity against subtilisin BPN' when the inhibitor was incubated with the enzyme in amounts less than one mol of enzyme per mol of the inhibitor. However, no inhibitor activity was observed when the molar ratio of the inhibitor to the enzyme was less than one. This phenomenon suggests that the carboxyl-terminal four amino acid residues might play an important role in the maintenance of the three-dimensional structure of SSI, which resists the action of the proteinase. The addition of more than 30-fold molar excess of SSI-(104-113)-decapeptide (C-terminal decapeptide of SSI) to the modified inhibitor resulted in refolding of the polypeptide chain, rendering it immune from proteolytic digestion.

Amino Acid Sequence↗

PubMed1981

Peptide bond synthesis catalyzed by subtilisin, papain, and pepsin.

The peptide bond syntheses catalyzed by subtilisin BPN', papain [EC 3.4.22.2], and pepsin [EC 3.4.23.1] were studied comparatively at the optimum pH of the enzymes with the coupling system Cbz-(AA)n-OH + Leu-X leads to Cbz-(AA)n-Leu-X, in which AA = various amino acid residues, n = 1-3 and X = -NH2, -OEt, -OBut, -ODPM (diphenyl methyl ester) or -NH. The coupling with these enzymes differed depending upon the nature of (AA)n and X. For subtilisin-catalyzed coupling, the molecular size of the carboxyl component was most important. Thus, Cbz-Gly-Pro-Leu-OH was useful for synthesis in the presence of an equimolar concentration of Leu-NH. Either Ala-NH2 or Leu-NH2 was also useful as an amine component when present at a concentration several times that of the carboxyl component. Papain catalyzed the coupling between Cbz-AA-OH (AA = glycine, L-alanine, L-valine, L-glutamic acid, or L-phenylalanine) and Leu-X (X = -OBut and -ODPM). The coupling of Cbz-Gly-Phe-OH and Leu-X catalyzed by pepsin was markedly affected, depending upon the nature of X in the following order: -NH greater than -OBut greater than -NH2, -OEt. Leu-NH2, however, was quite efficient when its molar concentration was raised to twenty times that of the carboxyl component.

Hydrogen-Ion Concentration↗

PubMed1997

Construction of novel subtilisin E with high specificity, activity and productivity through multiple amino acid substitutions.

Through three cumulative amino acid substitutions, we constructed novel mutant subtilisins E of Bacillus subtilis, all with high specificity, activity and productivity. The substitution of conserved Gly127, constituting P1 substrate-binding pocket, with Ala and Val showed a marked preference for the small P1 substrate. Leu was then substituted for Ile31 next to the catalytic Asp32 to enhance the catalytic activity. Both double mutants (I31L/G127A and I31L/G127V) showed a 3-5-fold increase in catalytic efficiency due to a large kcat, without any change in the specificity of the mutants at position 127. Molecular modeling suggests that large P1 residues were unable to access the pocket because of steric hindrance. A third mutation was introduced by replacing Tyr(-1) with Ala in the propeptide essential for autoprocessing to active mature subtilisin in vivo. A prominent 7-20-fold increase in active enzyme production occurred in the triple mutants (Y-1A/I31L/G127A and Y-1A/I31L/G127V).

Amino Acid Substitution↗

PubMed1994

Evidence for a large dispensable segment in the subtilisin-like catalytic domain of the Lactococcus lactis cell-envelope proteinase.

The Lactococcus lactis SK11 cell-envelope proteinase contains various inserts, located in external loops of the catalytic domain compared with related subtilisins. In this study, protein engineering was employed to determine the function of the largest loop insertion (residues 238-388) relative to the subtilisin structure. By site-directed mutagenesis we have deleted the fragment of the proteinase gene encoding these 151 residues and analyzed the mutant delta 238-388 proteinase for activity, (auto)processing and cleavage specificity. This extra segment is found to be inessential for activity, and its removal does not inhibit folding as the mutant proteinase is still active. In addition, the N- and C-terminal autoprocessing of the delta 238-388 proteinase appears to be unchanged. However, removal of residues 238-388 altered substantially the caseinolytic specificity of the enzyme, indicating that this extra segment influences substrate specificity. Residues 238-388 were shown to contain a specific epitope for a monoclonal antibody.

Amino Acid Sequence↗

PubMed2000

Characterization of a Casuarina glauca nodule-specific subtilisin-like protease gene, a homolog of Alnus glutinosa ag12.

In search of plant genes expressed during early interactions between Casuarina glauca and Frankia, we have isolated and characterized a C. glauca gene that has strong homology to subtilisin-like protease gene families of several plants including the actinorhizal nodulin gene ag12 of another actinorhizal plant, Alnus glutinosa. Based on the expression pattern of cg12 in the course of nodule development, it represents an early actinorhizal nodulin gene. Our results suggest that subtilisin-like proteases may be a common element in the process of infection of plant cells by Frankia in both Betulaceae (Alnus glutinosa) and Casuarinaceae (Casuarina glauca) symbioses.

Actinomycetales↗

PubMed1999

A novel member of the subtilisin-like protease family from Bacillus subtilis.

aprX is a 1326 bp gene of Bacillus subtilis strain 168 that encodes a serine protease, probably intracellular, characterized by significant similarity with subtilisins, thermitases and pyrolysins. Transcription analysis, performed by RT-PCR and primer extension, allowed the localization of the active promoter and showed that aprX is expressed in stationary phase. The pattern of expression of aprX and its dependence on various transition state regulatory genes (degU, degQ, hpr, abrB, sinR), monitored by lacZ transcriptional fusions, are distinctive from those of subtilisin and other degradative enzymes. aprX is not essential for either growth or sporulation.

Amino Acid Sequence↗

PubMed2000

Characterization of P69E and P69F, two differentially regulated genes encoding new members of the subtilisin-like proteinase family from tomato plants.

Subtilisin-like proteins represent an ancient family of serine proteases that are extremely widespread in living organisms. We report here the structure and genomic organization of two new transcriptionally active genes encoding proteins that belong to the P69 family of subtilisin-like proteases from tomato (Lycopersicon esculentum) plants. The two new members, P69E and P69F, are organized in a cluster and arranged in a tandem form. mRNA expression analysis and studies of transgenic Arabidopsis plants transformed with promoter-beta-glucuronidase fusions for each of these two genes revealed that they are differentially regulated, with each showing a highly specific mRNA expression pattern. P69E mRNA is expressed only in roots, while P69F mRNA is expressed only in hydathodes. A comparison of all the P69 amino acid sequences, gene structure, expression profiles, and clustered organization suggests a working model for P69 gene family evolution.

Amino Acid Sequence↗

PubMed1991

Complex between the subtilisin from a mesophilic bacterium and the leech inhibitor eglin-C.

The alkaline proteinase from the mesophilic bacterium Bacillus mesentericus has been crystallized in a 1:1 complex with the inhibitor eglin-C from the medical leech. The crystals have cell dimensions of a = 43.0, b = 71.9, c = 48.3 A and beta = 110.0 degrees and are in the space group P2(1). Three-dimensional data to 2.0 A have been recorded on film from a single crystal. The orientation and position of the complex in the unit cell have been established using the refined coordinates of subtilisin Carlsberg and of eglin-C as independent models. The structure of the complex has been refined by restrained least-squares minimization. The crystallographic R factor (= sigma[magnitude of Fo - magnitude of Fc[/sigma magnitude of Fo) is 15.1% including two Ca2+ ions and 312 water molecules. The structure is discussed in terms of its physicochemical properties in solution and its relation to other Bacillus subtilisins.

Amino Acid Sequence↗

PubMed1979

A method for designing peptide substrates for proteases. Tripeptidyl-p-nitroanilide substrates for subtilisin Carlsberg.

1. The kinetic parameters of 25 peptidyl-p-nitroanilide substrates were investigated with subtilisin Carlsberg as model enzyme. 2. For a series of 12 substrates, the contribution of various side chains to the affinity constant was computed by regression analysis. From these contributions the sequence of a new and better substrate, N-benzyloxycarbonyl-arginyl-norleucyl-norleucyl-p-nitroanilide (Z-Arg-Nle-Nle-Nan) was predicted. The compound was synthesized and assayed. Its calculated 1/Km value, 43.5 mM-1, was in a good agreement with the value of 40.0 mM-1 that was determined experimentally. 3. On expanding the series to 19 substrates, it was found that the productivity of enzyme-substrate binding is influenced primarily by those subsites which have a significantly greater contribution to the affinity constants than others. 4. The additivity principle applied reasonably well for the contribution of individual side chains to the kinetic parameters. This fact suggests that regression analysis can be used for the prediction of the amino acid sequence of better substrates than those already tested, probably not only for subtilisin but also for other proteolytic enzymes.

Kinetics↗

PubMed1992

Mutational replacements in subtilisin 309. Val104 has a modulating effect on the P4 substrate preference.

The previous notion that the amino acid side chain at position 104 of subtilisins is involved in the binding of the side chain at position P4 of the substrate has been investigated. The amino acid residue Val104 in subtilisin 309 has been replaced by Ala, Arg, Asp, Phe, Ser, Trp and Tyr by site-directed mutagenesis. It is shown that the P4 specificity of this enzyme is not determined solely by the amino acid residue occupying position 104, as the enzyme exhibits a marked preference for aromatic groups in P4, regardless of the nature of the position-104 residue. With hydrophilic amino acid residues at this position, no involvement is seen in binding of either hydrophobic or hydrophilic amino acid residues at position P4 of the substrates. The substrate with Asp in P4 is an exception, as the preference for this substrate is increased dramatically by introduction of an arginine residue at position 104 in the enzyme, presumably due to a substrate-induced conformational change. However, when position 104 is occupied by hydrophobic residues, it is highly involved in binding of hydrophobic amino acid residues, either by increasing the hydrophobicity of S4 or by determining the size of the pocket. The results suggest that the amino acid residue at position 104 is mobile such that it is positioned in the S4 binding site only when it can interact favourably with the substrate's side chain at position P4.

Amino Acid Sequence↗

PubMed1994

Homology modelling of the catalytic domain of human furin. A model for the eukaryotic subtilisin-like proprotein convertases.

A model is presented for the three-dimensional structure of the catalytic domain of the human serine proteinase furin and its interaction with model substrates. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin, and it also applies to other members of the eukaryotic subtilisin-like proprotein convertases. Predictions are made of the general protein fold, inserted loops, disulfide bonds, Ca(2+)-binding sites and salt bridges. A detailed prediction of the substrate-binding region attempts to explain the basis of specificity for multiple basic residues preceding the cleavage site. Specific acidic residues in the S1, S2 and S4 subsites of the substrate-binding region of furin are identified which appear to be of particular importance, while residues of the S2', S3, S5 and S6 subsites may also contribute to substrate binding. Based on this model, protein engineering can be employed not only to test the predicted enzyme-substrate interactions, as demonstrated for human furin, but, equally importantly, to design proprotein convertases with a desired specificity, or to design novel substrates or inhibitors.

Amino Acid Sequence↗

PubMed2006

Purification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1.

AIMS: The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. METHODS AND RESULTS: Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11.0. In BPP-A, zein was better substrate than casein at pH 13.0, whereas casein was better one than zein at pH 11.0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. CONCLUSION: A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates.

Amino Acid Sequence↗

PubMed2002

Improving the pathogenicity of a nematode-trapping fungus by genetic engineering of a subtilisin with nematotoxic activity.

Nematophagous fungi are soil-living fungi that are used as biological control agents of plant and animal parasitic nematodes. Their potential could be improved by genetic engineering, but the lack of information about the molecular background of the infection has precluded this development. In this paper we report that a subtilisin-like extracellular serine protease designated PII is an important pathogenicity factor in the common nematode-trapping fungus Arthrobotrys oligospora. The transcript of PII was not detected during the early stages of infection (adhesion and penetration), but high levels were expressed concurrent with the killing and colonization of the nematode. Disruption of the PII gene by homologous recombination had a limited effect on the pathogenicity of the fungus. However, mutants containing additional copies of the PII gene developed a higher number of infection structures and had an increased speed of capturing and killing nematodes compared to the wild type. The paralyzing activity of PII was verified by demonstrating that a heterologous-produced PII (in Aspergillus niger) had a nematotoxic activity when added to free-living nematodes. The toxic activity of PII was significantly higher than that of other commercially available serine proteases. This is the first report showing that genetic engineering can be used to improve the pathogenicity of a nematode-trapping fungus. In the future it should be possible to express recombinant subtilisins with nematicidal activity in other organisms that are present in the habitat of parasitic nematodes (e.g., host plant).

Animals↗

PubMed1989

Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain.

Alkaline elastase YaB is an extracellular serine protease of the alkalophilic Bacillus strain YaB. We cloned the structural gene, ale, and determined the nucleotide sequence. The mature enzyme (268 amino acids) was preceded by a putative signal sequence and a prosequence (27 and 83 amino acids, respectively). The mature enzyme was 55% homologous to subtilisin BPN'. Almost all the positively charged residues are predicted to be on the surface of the molecule, which would facilitate binding to elastin. The P1 substrate site-related sequences differed between alkaline elastase YaB and subtilisin BPN'.

Amino Acid Sequence↗

PubMed1994

In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly.

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.

Alleles↗