Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus.

The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 A resolution. The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg. The model of the PB92 protease has been refined to an R-factor of 14.0% and contains 1882 protein atoms, two calcium ions and 188 water molecules. The overall folding of the polypeptide chain closely resembles that of the subtilisins. Furthermore, almost all of the secondary structure elements found in subtilisin Carlsberg are also present in the PB92 protease. The major differences between the two structures are located around the deletion regions (residues 37 and 158-161 in subtilisin Carlsberg) and in two loops which are known to be the most variable parts of subtilisin structures. Flexibility of one of these loops (residues 126-130 in the PB92 protease) is believed to account for the induced-fit mechanism of substrate binding.     

Structural and mutational analyses of the interaction between the barley alpha-amylase/subtilisin inhibitor and the subtilisin savinase reveal a novel mode of inhibition

Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley α-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.     

Recombinant chymotrypsin inhibitor 2: expression, kinetic analysis of inhibition with alpha-chymotrypsin and wild-type and mutant subtilisin BPN', and protein engineering to investigate inhibitory specificity and mechanism

The serine protease inhibitor chymotrypsin inhibitor 2 (CI2 or BSPI2) has been expressed in Escherichia coli with the pINIIIompA3 expression vector to produce 20-40 mg/L of culture. Recombinant CI2 purified from this system has been characterized and found to be identical with CI2 from barley. Slow-binding kinetics were observed for the interaction between CI2 and subtilisin BPN', with Ki = 2.9 x 10(-12) M. Analysis of slow-binding data indicates that binding of the inhibitor follows the simplest model of E + I = EI with no kinetically detectable intermediate steps or proteolytic cleavage of the reactive site bond in CI2 (Met-59-Glu-60). This, in agreement with crystallographic data, indicates that the enzyme-inhibitor adduct is the Michaelis complex, which is not chemically processed by the enzyme. Three mutant CI2 molecules with new P1 residues have also been examined with a range of serine proteases, including a mutant subtilisin. In agreement with earlier studies, we find the P1 amino acid an important determinant of specificity. CI2 Met----Lys-59 was found to be a temporary inhibitor of subtilisin BPN' but an effective inhibitor of subtilisin Carlsberg and subtilisin BPN'(Glu----Ser-156). The structural reasons for this are discussed in relation to mechanisms of inhibition of serine proteases.     

Inhibitory specificity change of the ovomucoid third domain of the silver pheasant upon introduction of an engineered Cys14-Cys39 bond

The ovomucoid third domain from silver pheasant (OMSVP3), a typical Kazal-type inhibitor, strongly inhibits different serine proteases of various specificities, i.e., chymotrypsin, Streptomyces griseus protease, subtilisin, and elastase. Structural studies have suggested that conformational flexibility in the reactive site loop of the free inhibitor may be related to broad specificity of the ovomucoid. On the basis of the structural homology between OMSVP3 and ascidian trypsin inhibitor (ATI), which has a cystine-stabilized alpha-helical (CSH) motif in the sequence, we prepared the disulfide variant of OMSVP3, introducing an engineered disulfide bond between positions 14 and 39 near the reactive site (Met18-Glu19) by site-directed mutagenesis. The disulfide variant P14C/N39C retained potent inhibitory activities toward alpha-chymotrypsin (CHT) and S. griseus proteases A and B (SGPA and SGPB), while this variant lost most of its inhibitory activity toward porcine pancreatic elastase (PPE). We determined the solution structure of P14C/N39C, as well as that of wild-type OMSVP3, by two-dimensional nuclear magnetic resonance (2D NMR) methods and compared their structures to elucidate the structural basis of the inhibitory specificity change. For the molecular core consisting of a central alpha-helix and a three-stranded antiparallel beta-sheet, essentially no structural difference was detected between the two (pairwise rmsd value = 0.41 A). In contrast to this, a significant difference was detected in the loop from Cys8 to Thr17, where in P14C/N39C it has drawn approximately 4 A nearer the central helix to form the engineered Cys14-Cys39 bond. Concomitantly, the Tyr11-Pro12 cis-peptide linkage, which is highly conserved in ovomucoid third domains, was isomerized to the trans configuration. Such structural change in the loop near the reactive site may possibly affect the inhibitory specificity of P14C/N39C for the corresponding proteases.     

Characterization of structural determinants of granzyme B reveals potent mediators of extended substrate specificity

Granzymes are trypsin-like serine proteases mediating apoptotic cell death that are composed of two genetically distinct subfamilies: granzyme A-like proteases resemble trypsin in their active site architecture, while granzyme B-like proteases are quite distinct. Granzyme B prefers substrates containing P4 to P1 amino acids Ile/Val, Glu/Met/Gln, Pro/Xaa, and aspartic acid N-terminal to the proteolytic cleavage. By investigating the narrow extended specificity of the granzyme B-like proteases the mediators of their unique specificity are being defined. The foci of this study were the structural determinants Ile99, Tyr174, Arg192, and Asn218. Even modest mutations of these residues resulted in unique extended specificity profiles as determined using combinatorial substrate libraries and individual fluorogenic substrates. As with other serine proteases, Ile99 completely defines and predicts P2 specificity, primarily through the binding constant Km. Asn218 variants have minor effects alone but in combination with mutations at Arg192 and Ile99 alter P2 through P4 extended specificity. For each variant, the activity on its cognate substrate was equal to that of granzyme B for the same substrate. Thus, mutations at these determinants change extended selectivity preferentially over catalytic power. Additionally Asn218 variants result in increased activity on the wild type substrate, while the N218A/I99A variant disrupts the additivity between P2 and P4 specificity. This defines Asn218 not only as a determinant of specificity but also as a structural component required for P2 and P4 independence. This study confirms four determinants of granzyme B extended substrate specificity that constitute a canon applicable to the study of the remaining family members.     

Furin: the prototype mammalian subtilisin-like proprotein-processing enzyme. Endoproteolytic cleavage at paired basic residues of proproteins of the eukaryotic secretory pathway.

Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC 3.4.21.61), which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated. Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor.     

Enzyme-substrate interactions in the hydrolysis of peptide substrates by thermitase, subtilisin BPN', and proteinase K

Peptide substrates of the general structure acetyl-Alan (n = 2-5), acetyl-Pro-Ala-Pro-Phe-Alan-NH2 (n = 0-3), and acetyl-Pro-Ala-Pro-Phe-AA-NH2 (AA = various amino acids) were synthesized and used to investigate the enzyme-substrate interactions of the microbial serine proteases thermitase, subtilisin BPN', and proteinase K on the C-terminal side of the scissile bond. The elongation of the substrate peptide chain up to the second amino acid on the C-terminal side (P'2) enhances the hydrolysis rate of thermitase and subtilisin BPN', whereas for proteinase K an additional interaction with the third amino acid (P'3) is possible. The enzyme subsite S'1 specificity of the proteases investigated is very similar. With respect to kcat/Km values small amino acid residues such as Ala and Gly are favored in this position. Bulky residues such as Phe and Leu were hydrolyzed to a lower extent. Proline in P'1 abolishes the hydrolysis of the substrates. Enzyme-substrate interactions on the C-terminal side of the scissile bond appear to affect kcat more than Km for all three enzymes.     

Subtilisins of Bacillus spp. hydrolyze keratin and allow growth on feathers

Keratinase is a serine protease produced by Bacillus licheniformis PWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficient B. subtilis strain. Studies presented herein demonstrate that B. licheniformis Carlsberg strain NCIMB 6816, which produces the well-characterized serine protease subtilisin Carlsberg, also degrades and grows on feathers. The PWD-1 and Carlsberg strains showed a similar time-course of enzyme production, and the purified serine proteases have similar enzymatic properties on insoluble azokeratin and soluble FITC-casein. Kinetic analysis of both enzymes demonstrated that they have high specificity for aromatic and hydrophobic amino acids in the P1 substrate position, although keratinase discriminates more than subtilisin Carlsberg against charged residues at this site. Nucleotide sequence analysis of the serine protease genes from B. licheniformis strains PWD-1, Carlsberg NCIMB 6816, ATCC 12759, and NCIMB 10689 showed that the kerA-encoded protease of PWD-1 differs from the others only by having V222, rather than A222, near the active site serine S220. Further, high-level expression of subE-encoded subtilisin from B. subtilis (78% similar to subtilisin Carlsberg) also confers growth on feathers on a protease-deficient B. subtilis strain. While strain PWD-1 and the kerA protease efficiently degrade keratin, keratin hydrolysis and growth on feathers is a property that can be conferred by appropriate expression of the major subtilisins, including the industrially produced enzymes.     

Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene.

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.     

Cloning, characterization, and multiple chromosomal integration of a Bacillus alkaline protease gene

Extracellular Bacillus proteases are used as additives in detergent powders. We identified a Bacillus strain that produces a protease with an extremely alkaline pH optimum; this protease is suitable for use in modern alkaline detergent powders. The alkalophilic strain Bacillus alcalophilus PB92 gene encoding this high-alkaline serine protease was cloned and characterized. Sequence analysis revealed an open reading frame of 380 amino acids composed of a signal peptide (27 amino acids), a prosequence (84 amino acids), and a mature protein of 269 amino acids. Amino acid comparison with other serine proteases shows good homology with protease YaB, which is also produced by an alkalophilic Bacillus strain. Both show moderate homology with subtilisins but show some remarkable differences from subtilisins produced by neutrophilic bacilli. The prosequence of PB92 protease has no significant homology with prosequences of subtilisins. The abundance of negatively charged residues in the prosequences of PB92 protease is especially remarkable. The cloned gene was used to increase the production level of the protease. For this purpose the strategy of gene amplification in the original alkalophilic Bacillus strain was chosen. When introduced on a multicopy plasmid, the recombinant strain was unstable; under production conditions, plasmid segregation occurred. More stable ways of gene amplification were obtained by chromosomal integration. This was achieved by (i) homologous recombination, resulting in a strain with two tandemly arranged genes, and (ii) illegitimate recombination, resulting in a strain with a second copy of the protease gene on a locus not adjacent to the originally present gene. Both strains showed increased production and were more stable than the plasmid-containing strain. Absolute stability was only found when nontandem duplication occurred. This method of gene amplification circumvents stability problems often encountered in gene amplification in Bacillus species when plasmids or tandemly arranged genes in the chromosome are used.     

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.     

Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold

Evidence is presented, based on sequence comparison and secondary structure prediction, of structural and evolutionary relationship between chymotrypsin-like serine proteases, cysteine proteases of positive strand RNA viruses (3C proteases of picornaviruses and related enzymes of como-, nepo- and potyviruses) and putative serine protease of a sobemovirus. These observations lead to re-identification of principal catalytic residues of viral proteases. Instead of the pair of Cys and His, both located in the C-terminal part of 3C proteases, a triad of conserved His, Asp(Glu) and Cys(Ser) has been identified, the first two residues resident in the N-terminal, and Cys in the C-terminal beta-barrel domain. These residues are suggested to form a charge-transfer system similar to that formed by the catalytic triad of chymotrypsin-like proteases. Based on the structural analogy with chymotrypsin-like proteases, the His residue previously implicated in catalysis, together with two partially conserved Gly residues, is predicted to constitute part of the substrate-binding pocket of 3C proteases. A partially conserved ThrLys/Arg dipeptide located in the loop preceding the catalytic Cys is suggested to confer the primary cleavage specificity of 3C toward Glx/Gly(Ser) sites. These observations provide the first example of relatedness between proteases belonging, by definition, to different classes.     

Use of a structural alphabet to find compatible folds for amino acid sequences

The structural annotation of proteins with no detectable homologs of known 3D structure identified using sequence‐search methods is a major challenge today. We propose an original method that computes the conditional probabilities for the amino‐acid sequence of a protein to fit to known protein 3D structures using a structural alphabet, known as “Protein Blocks” (PBs). PBs constitute a library of 16 local structural prototypes that approximate every part of protein backbone structures. It is used to encode 3D protein structures into 1D PB sequences and to capture sequence to structure relationships. Our method relies on amino acid occurrence matrices, one for each PB, to score global and local threading of query amino acid sequences to protein folds encoded into PB sequences. It does not use any information from residue contacts or sequence‐search methods or explicit incorporation of hydrophobic effect. The performance of the method was assessed with independent test datasets derived from SCOP 1.75A. With a Z‐score cutoff that achieved 95% specificity (i.e., less than 5% false positives), global and local threading showed sensitivity of 64.1% and 34.2%, respectively. We further tested its performance on 57 difficult CASP10 targets that had no known homologs in PDB: 38 compatible templates were identified by our approach and 66% of these hits yielded correctly predicted structures. This method scales‐up well and offers promising perspectives for structural annotations at genomic level. It has been implemented in the form of a web‐server that is freely available at http://www.bo‐protscience.fr/forsa .     

Proline versus charge concept for protein stabilization against proteolytic attack

The virtue of the so-called 'proline concept' and the 'charge concept' for stabilizing protease-susceptible regions of a protein structure was compared on bovine pancreatic ribonuclease A. Alanine 20 and serine 21, both of which are located in a loop that is susceptible to the unspecific proteases subtilisin Carlsberg, subtilisin BPN', proteinase K and elastase, were replaced with proline or lysine by site-directed mutagenesis. The rate constant of proteolysis was decreased by up to three orders of magnitude for the proline mutants depending on the site of the mutation and the protease used. In contrast, substitution by lysine increased the proteolytic resistance by only one order of magnitude characterizing the 'proline concept' as superior to the 'charge concept'. Although the four applied proteases are considered to be unspecific, the degree of stabilization of the ribonuclease molecule varied considerably, indicating the impact of individual differences in their substrate specificity on the proteolytic resistance and degradation pathway of the target protein.     

One-step site-directed mutagenesis of the Kex2 protease oxyanion hole

BACKGROUND: Members of the subtilisin family of serine proteases usually have a conserved asparagine residue that stabilizes the oxyanion transition state of peptide-bond hydrolysis. Yeast Kex2 protease is a member of the subtilisin family that differs from the degradative subtilisin proteases in its high substrate specificity, it processes pro-alpha-factor, the precursor of the alpha-factor mating pheromone of yeast, and also removes the pro-peptide from its own precursor by an intramolecular cleavage reaction. Curiously, the mammalian protease PC2, a Kex2 homolog that is likely to be required for pro-insulin processing, has an aspartate in place of asparagine at the 'oxyanion hole'. RESULTS: We have tested the effect of making substitutions of the conserved oxyanion-hole asparagine (Asn 314) of the Kex2 protease. To do this, we have developed a rapid method of site-directed mutagenesis, involving homologous recombination of a polymerase chain reaction product in yeast. Using this method, we have substituted alanine or aspartate for Asn 314 in a form of Kex2 engineered for secretion. Transformants expressing the two mutant enzymes could be identified by failure either to produce mature alpha-factor or to mate. The Ala 314 enzyme was unstable but the Asp 314 enzyme accumulated to a high level, so that it could be purified and its activity towards various substrates tested in vitro. We found that, with three peptides that are good substrates of wild-type Kex2, the k(cal) of the Asp 314 enzyme was reduced approximately 4500-fold and its K(M) approximately 4-fold, relative to the wild-type enzyme. For the peptide substrate corresponding to the cleavage site of pro-alpha-factor, however, k(cat) of the Asp 314 enzyme was reduced only 125-fold, while the K(m) was increased 3-fold. Despite its reduced catalytic activity, however, processing of the mutant enzyme in vivo - by the intramolecular cleavage that removes its amino-terminal pro-domain - occurs at an unchanged rate. CONCLUSIONS: The effects of the Asn 314-Asp substitution reveal contributions to the reaction specificity of the Kex2 protease of substrate residues amino-terminal to the pair of basic residues at the cleavage site. Aspartate at the oxyanion hole appears to confer k(caf) discrimination between substrates by raising the energy barrier for productive substrate binding: this may have implications for pro-insulin processing by the PC2 protease, which has an aspartate at the equivalent position. The rate of intramolecular cleavage of pro-Kex2 may be limited by a step other than catalysis, presumably protein folding.     

Positive selection dictates the choice between kinetic and thermodynamic protein folding and stability in subtilases

Subtilisin E (SbtE) is a member of the ubiquitous superfamily of serine proteases called subtilases and serves as a model for understanding propeptide-mediated protein folding mechanisms. Unlike most proteins that adopt thermodynamically stable conformations, the native state of SbtE is trapped into a kinetically stable conformation. While kinetic stability offers distinct functional advantages to the native state, the constraints that dictate the selection between kinetic and thermodynamic folding and stability remain unknown. Using highly conserved subtilases, we demonstrate that adaptive evolution of sequence dictates selection of folding pathways. Intracellular and extracellular serine proteases (ISPs and ESPs, respectively) constitute two subfamilies within the family of subtilases that have highly conserved sequences, structures, and catalytic activities. Our studies on the folding pathways of subtilisin E (SbtE), an ESP, and its homologue intracellular serine protease 1 (ISP1), an ISP, show that although topology, contact order, and hydrophobicity that drive protein folding reactions are conserved, ISP1 and SbtE fold through significantly different pathways and kinetics. While SbtE absolutely requires the propeptide to fold into a kinetically trapped conformer, ISP1 folds to a thermodynamically stable state more than 1 million times faster and independent of a propeptide. Furthermore, kinetics establish that ISP1 and SbtE fold through different intermediate states. An evolutionary analysis of folding constraints in subtilases suggests that observed differences in folding pathways may be mediated through positive selection of specific residues that map mostly onto the protein surface. Together, our results demonstrate that closely related subtilases can fold through distinct pathways and mechanisms, and suggest that fine sequence details can dictate the choice between kinetic and thermodynamic folding and stability.     

Protection of actin against proteolysis by complex formation with deoxyribonuclease I

G-actin bound to deoxyribonuclease I (DNase I) is resistant to digestion by trypsin and chymotrypsin. In the absence of DNase I, G-actin is cleaved by these proteases to yield a 33 500 molecular weight core protein which is not degraded further. The major sites of proteolytic action in the amino acid sequence of actin have been identified as being adjacent to residues arginine-62 and lysine-68 for trypsin and leucine-57 for chymotrypsin. These residues are rendered inaccessible to proteases in the buffer by complex formation with DNase I. Digestion of G-actin with pronase from Streptomyces griseus yields fragmentation patterns that are similar to those observed with trypsin and chymotrypsin. This is likely to be because the specificities of the major constituents of pronase resemble those of trypsin and chymotrypsin. Again, complex formation with DNase I protects the otherwise vulnerable bonds in actin against proteolysis. Incubation with subtilisin Carlsberg leads to complete digestion of G-actin. No subtilisin-resistant core protein accumulates during the incubation. Protection of G-actin when complexed to DNase I is less than complete in this case but still is significant. This is interpreted in terms of the broad specificity of subtilisin and the observed fragmentation pattern of free G-actin when treated with subtilisin.     

Cold-Active Serine Alkaline Protease from the Psychrotrophic Bacterium Shewanella Strain Ac10: Gene Cloning and Enzyme Purification and Characterization

The gene encoding serine alkaline protease (SapSh) of the psychrotrophic bacterium Shewanella strain Ac10 was cloned in Escherichia coli. The amino acid sequence deduced from the 2,442-bp nucleotide sequence revealed that the protein was 814 amino acids long and had an estimated molecular weight of 85,113. SapSh exhibited sequence similarities with members of the subtilisin family of proteases, and there was a high level of conservation in the regions around a putative catalytic triad consisting of Asp-30, His-65, and Ser-369. The amino acid sequence contained the following regions which were assigned on the basis of homology to previously described sequences: a signal peptide (26 residues), a propeptide (117 residues), and an extension up to the C terminus (about 250 residues). Another feature of SapSh is the fact that the space between His-65 and Ser-369 is approximately 150 residues longer than the corresponding spaces in other proteases belonging to the subtilisin family. SapSh was purified to homogeneity from the culture supernatant of E. coli recombinant cells by affinity chromatography with a bacitracin-Sepharose column. The recombinant SapSh (rSapSh) was found to have a molecular weight of about 44,000 and to be highly active in the alkaline region (optimum pH, around 9.0) when azocasein and synthetic peptides were used as substrates. rSapSh was characterized by its high levels of activity at low temperatures; it was five times more active than subtilisin Carlsberg at temperatures ranging from 5 to 15°C. The activation energy for hydrolysis of azocasein by rSapSh was much lower than the activation energy for hydrolysis of azocasein by the subtilisin. However, rSapSh was far less stable than the subtilisin.     

Amino acid sequence analysis of barley stripe mosaic viral proteins: tentative identification of viral serine proteases

Analysis of amino acid sequences of barley stripe mosaic virus (BSMV) proteins revealed the pentapeptide GDSGG, the sequence unique for catalytic centers of serine chymotrypsin-like proteases, in protein p14 encoded by open reading frame 4 of RNA beta. Computer-assisted comparisons revealed a statistically significant similarity between amino acid sequences of p14 and chymotrypsin-like proteases. The catalytic His and Asp residues tentatively identified in p14 together with the Ser residue of the GDSGG sequence, presumably, constitute the "catalytic triad" characteristic of chymotrypsin-like proteases. Based on these observations and on the presence of a potential N-proximal transmembrane domain in p14, this protein may be suggested to be a serine protease involved in processing of the replicase precursor within a membrane-bound replication complex of BSMV.     

Apicomplexan rhomboids have a potential role in microneme protein cleavage during host cell invasion

Apicomplexan parasites secrete transmembrane (TM) adhesive proteins as part of the process leading to host cell attachment and invasion. These microneme proteins are cleaved in their TM domains by an unidentified protease termed microneme protein protease 1 (MPP1). The cleavage site sequence (IA downward arrowGG), mapped in the Toxoplasma gondii microneme proteins TgMIC2 and TgMIC6, is conserved in microneme proteins of other apicomplexans including Plasmodium species. We report here the characterisation of novel T. gondii proteins belonging to the rhomboid family of intramembrane-cleaving serine proteases. T. gondii possesses six genes encoding rhomboid-like proteins. Four are localised along the secretory pathway and therefore constitute possible candidates for MPP1 activity. Toxoplasma rhomboids TgROM1, TgROM2 and TgROM5 cleave the TM domain of Drosophila Spitz, an established substrate for rhomboids from several species, demonstrating that they are active proteases. In addition, TgROM2 cleaves chimeric proteins that contain the TM domains of TgMIC2 and TgMIC12.