Modeling the 3D structure of wheat subtilisin/chymotrypsin inhibitor (WSCI). Probing the reactive site with two susceptible proteinases by time-course analysis and molecular dynamics simulations

Comparative modeling and time-course hydrolysis experiments have been applied to investigate two enzyme-inhibitor complexes formed between the wheat subtilisin-chymotrypsin inhibitor (WSCI) and two susceptible proteinases. WSCI represents the first case of a wheat protein inhibitor active against animal chymotrypsins and bacterial subtilisins. The model was created using as template structure that of the CI-2A inhibitor from barley (PDB code: 2CI2), which shares 87% sequence identity with WSCI. Under these conditions of high similarity, the comparative modeling approach can be successfully applied. We predicted the WSCI 3D model and used it to investigate enzyme-inhibitor complex systems. Experimental observations indicated that chymotrypsin, but not subtilisin, in addition to cleavage at the primary reactive site Met48-Glu49, is able to hydrolyze a second peptide bond between Phe58 and Val59. Here, we report on cleavage of the peptide bond at the inhibitor's reactive site (Met48-Glu49) determined using time-course hydrolysis experiments; the same event was investigated for both subtilisin/WSCI and chymotrypsin/WSCI complexes using molecular dynamics simulations. The molecular details of the initial inhibitor-enzyme interactions, as well as of the changes observed during the simulations, allow us to speculate on the different fates of the two WSCI-proteinase complexes.     

Primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin

The proteinase inhibitor WSCI, active in inhibiting bacterial subtilisin and a number of animal chymotrypsins, was purified from endosperm of exaploid wheat (Triticum aestivum, c.v. San Pastore) by ion exchange chromatography and its complete amino acid sequence was established by automated Edman degradation. WSCI consists of a single polypeptide chain of 72 amino acid residues, has a molecular mass of 8126.3 Da and a pl of 5.8. The inhibition constants (Ki) for Bacillus licheniformis subtilisin and bovine pancreatic alpha-chymotrypsin are 3.92 x 10(-9) M and 7.24 x 10(-9) M, respectively. The inhibitor contains one methionine and of tryptophan residue and has a high content of essential amino acids (41 over a total of 72 residues), but no cysteines. The primary structure of WSCI shows high similarity with barley subtilisin-chymotrypsin isoinhibitors of the Cl-2 type and with maize subtilisinchymotrypsin inhibitor MPI. Significant degrees of similarity were also found between sequences of WSCI and of other members of the potato inhibitor I family of the serine proteinase inhibitors. The wheat inhibitor WSCI has a single reactive site (the peptide bond between methionyl-48 and glutamyl-49 residues) as identified by affinity chromatography and sequence analysis.     

Redesigning the reactive site loop of the wheat subtilisin/chymotrypsin inhibitor (WSCI) by site-directed mutagenesis. A protein–protein interaction study by affinity chromatography and molecular modeling

A site-directed mutagenesis strategy was employed to obtain four mutants of wheat subtilisin/chymotrypsin inhibitor (WSCI), with the aim to produce inactive forms of this protein. The mutants were expressed in Escherichia coli as fusion proteins and, after the tag removal, were purified to homogeneity. Three mutants, containing a single mutation at the sequence positions 49 or 50, were named E49S, E49P and Y50G, respectively. These mutants exhibited anti-subtilisin activities comparable to that of the wild type protein; instead, anti-chymotrypsin activity was detectable only for the mutant E49S. A fourth mutant (M48P-E49G), containing a double amino acid substitution at the inhibitor reactive site (P1–P1′), was inactive against both subtilisin and chymotrypsin. In order to investigate the interactions between the putative susceptible enzymes and the mutated forms of WSCI, we performed time-course hydrolysis experiments by incubating samples of the mutants with subtilisin–agarose and chymotrypsin–agarose, respectively. These experiments yielded information on the E/I complex formation, as well as on the timing of the cleavage pattern of some of these mutants. Molecular modeling studies were carried out with the 3D models of the mutants and of their putative complexes with subtilisin and chymotrypsin. In terms of inter- and intra-chain H-bond networks, the observations made for each theoretical E/I complex were found to be fully coherent with experimental data (kinetic and time-course hydrolysis) and supplied specific modalities of interaction of each mutant with the enzyme counterpart.     

Design of a small peptide-based proteinase inhibitor by modeling the active-site region of barley chymotrypsin inhibitor 2

A synthetic peptide-based proteinase inhibitor was constructed by modeling the regions responsible for inhibition in barley chymotrypsin inhibitor 2 (CI-2). The 18-residue peptide was designed by molecular modeling, based on the crystal structure of CI-2. The amino acid sequences that interact with the proteinase were preserved, as well as residues that maintain the structure of the inhibitory loop. A disulfide bridge was introduced to force the peptide to adopt a cyclic structure. Kinetic studies on binding of the cyclic peptide to subtilisin BPN', subtilisin Carlsberg, chymotrypsin, and pancreatic elastase show that the cyclic peptide retains both the inhibition properties, the kinetic mechanism, and the specificity of the original protein inhibitor. Formation of a cyclic structure was found to be essential, and activity was abolished by reduction of the disulfide. As with CI-2, tightest binding is found to subtilisin BPN', where the Ki value for the cyclic peptide was 28 x 10(-12) M, compared with 29 x 10(-12)M for CI-2 under identical conditions. This remarkable result shows that it is possible to use a short synthetic peptide to model the molecular recognition properties of the intact protein, in this case obtaining full functionality with just 18 residues instead of 83 for CI-2.     

Purification and amino acid sequence of a bitter gourd inhibitor against an acidic amino acid-specific endopeptidase of Streptomyces griseus.

An inhibitor (BGIA) against an acidic amino acid-specific endopeptidase of Streptomyces griseus (Glu S. griseus protease) was isolated from seeds of the bitter gourd Momordica charantia L., and its amino acid sequence was determined. The molecular weight of BGIA based on the amino acid sequence was calculated to be 7419. BGIA competitively inhibited Glu S. griseus protease with an inhibition constant (Ki) of 70 nM, and gel filtration analyses suggested that BGIA forms a 1:1 complex with this protease. However, two other acidic amino acid-specific endopeptidases, protease V8 from Staphylococcus aureus and Bacillus subtilis proteinase (Glu B. subtilis protease), were not inhibited by BGIA. BGIA had no inhibitory activity against chymotrypsin, trypsin, porcine pancreatic elastase, and papain, although subtilisin Carlsberg was strongly inhibited. The amino acid sequence of BGIA shows similarity to potato chymotrypsin inhibitor, barley subtilisin-chymotrypsin inhibitor CI-1 and CI-2, and leech eglin C, especially around the reactive site. Although the residue at the putative reactive site of these inhibitors is leucine or methionine, the corresponding amino acid in BGIA is alanine.     

Isolation and characterization of a novel, developmentally regulated proteinase inhibitor I protein and cDNA from the fruit of a wild species of tomato

A novel member of the proteinase Inhibitor I family having a trypsin inhibitor specificity was isolated from the fruit of the wild tomato species Lycopersicon peruvianum (L.) Mill. (LA 107) and characterized. The protein is among the isoinhibitors of Inhibitor I that comprise 50% of the soluble proteins in the fruit of this wild species of tomato. A cDNA corresponding to the inhibitor protein and mRNA was isolated and characterized. The Inhibitor I mRNA represented 0.06% of the poly(A) RNA and gene copy number reconstruction experiments gave an estimate of two to four genes/haploid genome. The open reading frame of the cDNA codes for a protein of 111 amino acids having a 42-amino acid prepropolypeptide. The NH2-terminal sequence of the first 21 amino acids of the purified Inhibitor I protein confirmed that the cDNA was identical to the protein. The amino acid sequence of the L. peruvianum fruit Inhibitor I exhibits 74% identity with the wound-inducible Inhibitor I from tomato leaves. Whereas all previously identified members of the Inhibitor I family have either Met, Leu, or Asp at the P1 site and can inhibit enzymes such as chymotrypsin, subtilisin, and elastase, the fruit Inhibitor I possesses Lys at the P1 position. Thus, this is the first member of the extensive Inhibitor I family from plants and animals that exhibits trypsin inhibitory specificity. The presence of this inhibitor in wild tomato fruit may reflect a functional role to protect the tissues against herbivory.     

Localization to chromosomes of structural genes for the major protease inhibitors of barley grains

Summary Wheat-barley chromosome addition lines were compared by isoelectric focusing of protein extracts to identify chromosomes carrying loci for the major immunochemically distinct protease inhibitors of barley grains. Structural genes for the following inhibitors were localized: an inhibitor of both endogenous -amylase 2 and subtilisin (ASI) on chromosome 2, two chymotrypsin/subtilisin inhibitors (CI-1 and CI-2) on chromosome 5 (long arm) and the major trypsin inhibitor (TI-1) on chromosome 3.     

Differential synthesis in vitro of barley aleurone and starchy endosperm proteins

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA₃. B and C hordein polypeptides and the salt-soluble proteins β-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the α-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, β-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of α-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for α-amylase, these hormones have the opposite effect on ASI mRNA levels.     

Rice bifunctional alpha-amylase/subtilisin inhibitor: cloning and characterization of the recombinant inhibitor expressed in Escherichia coli

The complete nucleotide sequences of the cDNA and its gene that encode a bifunctional alpha-amylase/subtilisin inhibitor of rice (Oryza sativa L.) (RASI) were analyzed. RASI cDNA (939 bp) encoded a 200-residue polypeptide with a molecular mass of 21,417 Da, including a signal peptide of 22 amino acids. Sequence comparison and phylogenetic analysis showed that RASI is closely related to alpha-amylase/subtilisin inhibitors from barley and wheat. RASI was found to be expressed only in seeds, suggesting that it has a seed-specific function. A coding region of RASI cDNA without the signal peptide was introduced into Escherichia coli and was expressed as a His-tagged protein. Recombinant RASI was purified to homogeneity in a single step by Ni-chelating affinity column chromatography and characterized to elucidate the target enzyme. The recombinant inhibitor had strong inhibitory activity toward subtilisin, with an equimolar relationship, comparable with that of native RASI, and weak inhibitory activity toward some microbial alpha-amylases, but not toward animal or insect alpha-amylases. These results suggest that RASI might function in the defense of the seed against microorganisms.     

Molecular characterization of a gene encoding extracellular serine protease isolated from a subtilisin inhibitor-deficient mutant of Streptomyces albogriseolus S-3253.

An extracellular serine protease produced by a mutant, M1, derived from Streptomyces albogriseolus S-3253 that no longer produces a protease inhibitor (Streptomyces subtilisin inhibitor [SSI]) was isolated. A 20-kDa protein was purified by its affinity for SSI and designated SAM-P20. The amino acid sequence of the amino-terminal region of SAM-P20 revealed high homology with the sequences of Streptomyces griseus proteases A and B, and the gene sequence confirmed the relationships. The sequence also revealed a putative amino acid signal sequence for SAM-P20 that apparently functioned to allow secretion of SAM-P20 from Escherichia coli carrying the recombinant gene. SAM-P20 produced by E. coli cells was shown to be sensitive to SSI inhibition.     

Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus

An Aspergillus fumigatus extracellular alkaline protease (ALP) which is an enzyme of the subtilisin family is a potential virulent factor of the fungus. The gene encoding ALP was isolated from a genomic library made from DNA of an A. fumigatus isolate. The nucleotide sequence of this gene was compared to that of a cDNA encoding A. oryzae ALP and to that of a cDNA from A. fumigatus encoding the mature ALP protein. Mature A. fumigatus ALP contains 282 amino acids and is encoded by three exons. The pre-proenzyme has a leader sequence of 121 amino acids.     

Isolation of a cDNA encoding a granule-bound 152-kilodalton starch-branching enzyme in wheat

Screening of a wheat (Triticum aestivum) cDNA library for starch-branching enzyme I (SBEI) genes combined with 5'-rapid amplification of cDNA ends resulted in isolation of a 4,563-bp composite cDNA, Sbe1c. Based on sequence alignment to characterized SBEI cDNA clones isolated from plants, the SBEIc predicted from the cDNA sequence was produced with a transit peptide directing the polypeptide into plastids. Furthermore, the predicted mature form of SBEIc was much larger (152 kD) than previously characterized plant SBEI (80-100 kD) and contained a partial duplication of SBEI sequences. The first SBEI domain showed high amino acid similarity to a 74-kD wheat SBEI-like protein that is inactive as a branching enzyme when expressed in Escherichia coli. The second SBEI domain on SBEIc was identical in sequence to a functional 87-kD SBEI produced in the wheat endosperm. Immunoblot analysis of proteins produced in developing wheat kernels demonstrated that the 152-kD SBEIc was, in contrast to the 87- to 88-kD SBEI, preferentially associated with the starch granules. Proteins similar in size and recognized by wheat SBEI antibodies were also present in Triticum monococcum, Triticum tauschii, and Triticum turgidum subsp. durum.     

Crystal and molecular structure of the inhibitor eglin from leeches in complex with subtilisin Carlsberg

The crystal structure of the molecular complex of eglin, a serine proteinase inhibitor from leeches, with subtilisin Carlsberg has been determined at 2.0 A resolution by the molecular replacement method. The complex has been refined by restrained-parameter least-squares. The present crystallographic R factor (Formula: see text) is 0.183. Eglin is a member of the potato inhibitor 1 family, a group of serine proteinase inhibitors lacking disulfide bonds. Eglin shows strong structural homology to CI-2, a related inhibitor from barley seeds. The structure of subtilisin Carlsberg in this complex is very similar to the known structure from barley seeds. The structure of subtilisin Carlsberg in this complex is very similar to the known structure of subtilisin novo, despite changes of 84 out of 274 amino acids.     

The amino acid sequence of a 20 kDa bifunctional subtilisin/alpha-amylase inhibitor from bran [correction of brain] of rice (Oryza sativa L.) seeds.

A 20 kDa bifunctional inhibitor of the microbial proteinase, subtilisin, and the alpha-amylase from the larvae of the red flour beetle (Tribolium castaneum) was purified from bran of rice seeds by saline extraction, precipitation with ammonium sulphate, ion-exchange chromatography on DEAE-Cellulose and Toyopearl CM-650, and preparative HPLC on Vydac C18. The complete primary structure was determined by automatic degradation of the intact, reduced and S-alkylated protein, and by manual DABITC/PITC micro-sequencing of peptides obtained from the protein following separate enzymic digestions with trypsin, pepsin, chymotrypsin, elastase and the protease from S. aureus V8. The protein sequence, which contained 176 residues, showed strong homology with similar bifunctional inhibitors previously isolated from wheat and barley which are related to the Kunitz family of proteinase inhibitors from legume seeds.     

Properties and Specific Functional Features of Wheat Grain α-Amylase/Subtilisin Inhibitor

A protein bifunctional inhibitor of endogenous α-amylase and subtilisin has been isolated from wheat grain and purified. The inhibitor specifically inactivates α-amylase isozymes with high isoelectric point values (group α-AMY1) and has almost no effect on the α-AMY2 isozymes with low isoelectric point values. This enzyme does not belong to glycoproteins and has a molecular weight of 21 kDa and an isoelectric point of 7.2. The protein displays a relatively high thermostability and pH optimum of 8.0; its inhibitory activity requires the presence of Ca²⁺ cations. The inhibition of excess α-amylase in wheat grain with a low falling number by the purified protein is studied.     

A novel slow-tight binding serine protease inhibitor from eastern oyster (Crassostrea virginica) plasma inhibits perkinsin, the major extracellular protease of the oyster protozoan parasite Perkinsus marinus

A serine protease inhibitor was purified from plasma of the eastern oyster, Crassostrea virginica. The inhibitor is a 7609.6 Da protein consisting of 71 amino acids with 12 cysteine residues that are postulated to form 6 intra-chain disulfide bridges. Sequencing of the cloned cDNA identified an open reading frame encoding a polypeptide of 90 amino acids, with the 19 N-terminal amino acids forming a signal peptide. No sequence similarity with known proteins was found in sequence databases. The protein inhibited the serine proteases subtilisin A, trypsin and perkinsin, the major extracellular protease of the oyster protozoan parasite, Perkinsus marinus, in a slow binding manner. The mechanism of inhibition involves a rapid binding of inhibitor to the enzyme to form a weak enzyme-inhibitor complex followed by a slow isomerization to form a very tight binding enzyme-inhibitor complex. The overall dissociation constants K(i) with subtilisin A, perkinsin and trypsin were 0.29 nM, 13.7 nM and 17.7 nM, respectively. No inhibition of representatives of the other protease classes was detected. This is the first protein inhibitor of proteases identified from a bivalve mollusk and it represents a new protease inhibitor family. Its tight binding to subtilisin and perkinsin suggests it plays a role in the oyster host defense against P. marinus.     

Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened from Doen-Jang

Bacillus sp. strain DJ-4, which produces extracellular proteases, was screened from Doen-Jang, a traditional Korean fermented food. A fibrinolytic enzyme (subtilisin DJ-4) was purified using commercial chromatographic techniques. The relative molecular mass of the isolated protein was 29 kDa by SDS-PAGE and fibrin zymography assay. The enzyme was characterized as a serine protease by an inhibitor assay on the fibrin zymography gel and by an amidolytic assay using a chromogenic substrate. The enzyme was inhibited by PMSF, but not by EDTA or leupeptin. The first 14 amino acids of the N-terminal sequence were identical to that of subtilisin BPN', but the activity of subtilisin DJ-4 was 2.2 and 4.3 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively.