Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M _r) of 28.7 kDa, whereas protease B, with a M _r of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K _m values of these two proteases on SAAPF-pNa were higher than that for α-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH₂-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family. Journal of Industrial Microbiology & Biotechnology (2001) 26, 387–393. Received 05 November 2000/ Accepted in revised form 23 April 2001     

Inhibitors of chymotrypsin and microbial serine proteases in barley grains

Barley grains contain two imrnunochemically distinct inhibitors of chymotrypsin and microbial serine proteases. Both inhibitors are rich in lysine (9.5 and 11.5 g Lys/g protein). Hiproly high-lysine barley contains twenty-fold higher, high-lysine mutant 1508 five-fold higher amounts of these inhibitors than normally cultivated varieties. Inhibitors were extracted from Hiproly barley, and ammonium sulfate fractionation followed by gel filtration resulted in a neariy complete separation of the two inhibitors. No inactive protein impurities could be detected in a number of isoinhibitor preparations obtained in subsequent cation exchange chrotnatography steps. One inhibitor (CI-1) was composed of at leas# 4 molecular forms with isoelecfric points in the range 4.75–5.55 and a monomer molecular size of about 9 000. Most of this inhibitor was apparently present as dimer forms in grain extracts. The other inhibitor (CI-2) included at least 7 different molecular forms with isoelectric points in the range 6.05–7.90 and different molecular sizes in the range 6 500–9 000. Both dimer and monomer forms were present in grain extracts. In contrast to previously purified protease inhibitors of plant origin, the two barley inhibitors contain no cysteine. No interactions between the two inhibitors and trypsin were observed, but the inhibitors were immediately inactivated by pepsin at pH 2.0. Monospecific antibodies towards the two inhibitors were obtained after immunization with glutaraldehyde-polymerized inhibitor.
Inhibitor CI-1 is identical with an inhibitor of microbial alkaline proteases previously purified (Mikola and Suolinna 1971. Arch. Biochem. Biophys. 144: 566–575).     

Cleavage specificity of the serine protease of Aeromonas sobria, a member of the kexin family of subtilases

Subtilisin-like proteases have been grouped into six families based on a sequence of the catalytic domain. One of the six is the kexin family, of which furin is a representative protease. All members of the kexin family, except one, are from eukaryotes. The one prokaryotic protease is a serine protease of Aeromonas sorbria (ASP). Here, we examined the substrate specificity of ASP based on the cleavage of short peptides. The results showed that ASP preferentially cleaves the peptide bond following two basic residues, one of which is Lys, but not the bond following a single basic residue. This indicates that the tertiary structure around the catalytic domain of ASP resembles, but is not identical to that of furin. Prekallikrein was cleaved into four fragments by ASP, indicating that the protein must be cleaved at specific sequences.     

Modeling and structural analysis of evolutionarily diverse S8 family serine proteases

Serine proteases are an abundant class of enzymes that are involved in a wide range of physiological processes and are classified into clans sharing structural homology. The active site of the subtilisin-like clan contains a catalytic triad in the order Asp, His, Ser (S8 family) or a catalytic tetrad in the order Glu, Asp and Ser (S53 family). The core structure and active site geometry of these proteases is of interest for many applications. The aim of this study was to investigate the structural properties of different S8 family serine proteases from a diverse range of taxa using molecular modeling techniques. In conjunction with 12 experimentally determined three-dimensional structures of S8 family members, our predicted structures from an archaeon, protozoan and a plant were used for analysis of the catalytic core. Amino acid sequences were obtained from the MEROPS database and submitted to the LOOPP server for threading based structure prediction. The predicted structures were refined and validated using PROCHECK, SCRWL and MODELYN. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S8 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, the analysis presented here is beneficial for future molecular modeling strategies and structure-based rational drug design.     

ClpP: a distinctive family of cylindrical energy-dependent serine proteases

Processes maintaining protein homeostasis in the cell are governed by the activities of molecular chaperones that mainly assist in the folding of polypeptide chains and by a large class of proteases that regulate protein levels through degradation. ClpP proteases define a distinctive family of cylindrical, energy-dependent serine proteases that are highly conserved throughout bacteria and eukaryota. They typically interact with ATP-dependent AAA+ chaperones that bind and unfold target substrates and then translocate them into ClpP for degradation. Structural and functional studies have provided a detailed view of the mechanism of function of this class of proteases.     

The structural basis for catalysis and substrate specificity of a rhomboid protease

Rhomboids are intramembrane proteases that use a catalytic dyad of serine and histidine for proteolysis. They are conserved in both prokaryotes and eukaryotes and regulate cellular processes as diverse as intercellular signalling, parasitic invasion of host cells, and mitochondrial morphology. Their widespread biological significance and consequent medical potential provides a strong incentive to understand the mechanism of these unusual enzymes for identification of specific inhibitors. In this study, we describe the structure of Escherichia coli rhomboid GlpG covalently bound to a mechanism‐based isocoumarin inhibitor. We identify the position of the oxyanion hole, and the S₁‐ and S₂′‐binding subsites of GlpG, which are the key determinants of substrate specificity. The inhibitor‐bound structure suggests that subtle structural change is sufficient for catalysis, as opposed to large changes proposed from previous structures of unliganded GlpG. Using bound inhibitor as a template, we present a model for substrate binding at the active site and biochemically test its validity. This study provides a foundation for a structural explanation of rhomboid specificity and mechanism, and for inhibitor design.     

Characterization of a keratinolytic protease produced by the feather-degrading Amazonian bacterium Bacillus sp. P45

Abstract

An extracellular keratinolytic protease produced by Bacillus sp. P45 was purified and characterized. The keratinase had a molecular weight of approximately 26 kDa and was active over wide pH and temperature ranges, with optimal activity at 55°C and pH 8.0. However, this enzyme displayed low thermostability, being completely inactivated after 10 min at 50°C. Keratinase activity increased with Ca²⁺, Mg²⁺, Triton X-100, ethanol and DMSO, was stable in the presence of the reducing agent 2-mercaptoethanol, and was inactivated by SDS. PMSF (phenylmethylsulfonyl fluoride) completely inactivated and EDTA strongly inhibited the enzyme, indicating that the keratinase is a serine protease depending on metal ions for optimal activity and/or stability. Accordingly, analysis of tryptic peptides revealed sequence homologies which characterize the keratinase as a subtilisin-like serine protease. The purified enzyme was able to hydrolyze azokeratin and keratin azure. Casein was hydrolyzed at higher rates than keratinous substrates, and 2-mercaptoethanol tended to enhance keratin hydrolysis. With synthetic substrates, the keratinase showed a preference for aromatic and hydrophobic residues at the P1 position of tetrapeptides; the enzyme was not active, or the activity was drastically diminished, towards shorter peptides. Keratinase from Bacillus sp. P45 might potentially be employed in the production of protein hydrolysates at moderate temperatures, being suitable for the bioconversion of protein-rich wastes through an environmentally friendly process requiring low energy inputs.     

Three serine proteases from the latex of Euphorbia cyparissias

Three serine-centred proteolytic enzymes, euphorbains y-1, ?2 and ?3, were isolated from the latex of Euphorbia cyparissias. These proteases have different specific activities to azocoll and CBZ glycine p-nitrophenyl ester. The pIs and M_rs of y-1, ?2 and ?3 are 5.2 and 67 000, 5.2 and 33 000, and 6.3 and 67 000, respectively. The enzymes, which are glycoproteins, are immunologically distinct from euphorbain 1, but clearly related to that enzyme in amino acid composition.     

A conserved element that stabilizes the group II intron active site

The internal loop at the base of domain 3 (D3) is one of the most conserved and catalytically important elements of a group II intron. However, the location and molecular nature of its tertiary interaction partners has remained unknown. By employing a combination of site-directed photo-cross-linking and nucleotide analog interference suppression (NAIS), we show that the domain 3 internal loop (D3IL) interacts with the epsilon-epsilon' duplex, which is an active-site element located near the 5'-splice site in D1. Our data also suggest that the D3IL may interact with the bulge of D5, which is a critical active site component. The results of this and other recent studies indicate that the D3IL participates in a complex network of tertiary interactions involving epsilon-epsilon', the bulge of D5 and J23, and that it helps to optimize active site architecture by supporting interactions among these catalytic motifs. Our results are consistent with the role of D3 as a catalytic effector that enhances intron reactivity through active site stabilization.     

Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family

Ezrin, previously also known as cytovillin, p81, and 80K, is a cytoplasmic protein enriched in microvilli and other cell surface structures. Ezrin is postulated to have a membrane-cytoskeleton linker role. Recent findings have also revealed that the NH2-terminal domain of ezrin is associated with the plasma membrane and the COOH-terminal domain with the cytoskeleton (Algrain, M., O. Turunen, A. Vaheri, D. Louvard, and M. Arpin. 1993. J. Cell Biol. 120: 129-139). Using bacterially expressed fragments of ezrin we now demonstrate that ezrin has an actin-binding capability. We used glutathione-S-transferase fusion proteins of truncated ezrin in affinity chromatography to bind actin from the cell extract or purified rabbit muscle actin. We detected a binding site for filamentous actin that was localized to the COOH-terminal 34 amino acids of ezrin. No binding of monomeric actin was detected in the assay. The region corresponding to the COOH- terminal actin-binding site in ezrin is highly conserved in moesin, actin-capping protein radixin and EM10 protein of E. multilocularis, but not in merlin/schwannomin. Consequently, this site is a potential actin-binding site also in the other members of the protein family. Furthermore, the actin-binding site in ezrin shows sequence homology to the actin-binding site in the COOH terminus of the beta subunit of the actin-capping protein CapZ and one of the potential actin-binding sites in myosin heavy chain. The actin-binding capability of ezrin supports its proposed role as a membrane-cytoskeleton linker.     

Structure and energetics of protein-protein interactions: the role of conformational heterogeneity in OMTKY3 binding to serine proteases

Proteins with flexible binding surfaces can interact with numerous binding partners. However, this promiscuity is more difficult to understand in "rigid-body" proteins, whose binding results in little, or no, change in the position of backbone atoms. The binding of Kazal inhibitors to serine proteases is considered a classic case of rigid-body binding, although they bind to a wide range of proteases. We have studied the thermodynamics of binding of the Kazal serine protease inhibitor, turkey ovomucoid third domain (OMTKY3), to the serine protease subtilisin Carlsberg using isothermal titration calorimetry and have determined the crystal structure of the complex at very high resolution (1.1A). Comparison of the binding energetics and structure to other OMTKY3 interactions demonstrates that small changes in the position of side-chains can make significant contributions to the binding thermodynamics, including the enthalpy of binding. These effects emphasize that small, "rigid-body" proteins are still dynamic structures, and these dynamics make contributions to both the enthalpy and entropy of binding interactions.     

Comparison of substrate specificities against the fusion glycoprotein of virulent Newcastle disease virus between a chick embryo fibroblast processing protease and mammalian subtilisin-like proteases

The fusion (F) protein precursor of virulent Newcastle disease virus (NDV) strains has two pairs of basic amino acids at the cleavage site, and its intracellular cleavage activation occurs in a variety of cells; therefore, the viruses cause systemic infections in poultry. To explore the protease responsible for the cleavage in the natural host, we examined detailed substrate specificity of the enzyme in chick embryo fibroblasts (CEF) using a panel of the F protein mutants at the cleavage site expressed by vaccinia virus vectors, and compared the specificity with those of mammalian subtilisin-like proteases such as furin, PC6 and PACE4 which are candidates for F protein processing enzymes. It was demonstrated in CEF cells that Arg residues at the -4, -2 and -1 positions upstream of the cleavage site were essential, and that at the -5 position was required for maximal cleavage. Phe at the +1 position was also important for efficient cleavage. On the other hand, furin and PC6 expressed by vaccinia virus vectors showed cleavage specificities against the F protein mutants consistent with that shown by the processing enzyme of CEF cells, but PACE4 hardly cleaved the F proteins including the wild type. These results indicate that the proteolytic processing enzymes of poultry for virulent NDV F proteins could be furin and/or PC6 but not PACE4. The significance of individual contribution of the three amino acids at the -5, -2 and +1 positions to cleavability was discussed in relation to the evolution of virulent and avirulent NDV strains.     

Characterization of the complex between mannose-binding lectin trimer and mannose-binding lectin-associated serine proteases

Mannose-binding lectin (MBL) is an oligomeric serum lectin involved in innate immunity. Human MBL is complexed with three types of serine proteases (MASP-1, MASP-2 and MASP-3) and two types of their truncated forms (sMAP and MAp44). When an MBL complex binds to carbohydrates of pathogens, the complement system is activated via the lectin pathway. Human MBL is a mixture of different sized oligomers that range mainly from trimers to hexamers. It has been suggested that different MBL oligomers may have distinct MASP compositions. In the present study, an MBL trimer (MBL-I) exclusive of other oligomers was isolated from human serum by chromatography. Immunoblot analysis of MBL-I revealed that it had been co-purified with MASP-1 and sMAP. This suggests that MASP-1 and sMAP are bound to each other in MBL-I. The MBL-I complex was found to activate C2, but to lack the ability to activate C4 due to the absence of MASP-2.     

The influence of residue 190 in the S1 site of trypsin-like serine proteases on substrate selectivity is universally conserved

We examined the influence of Ser/Ala190 in the S1 site on P1 substrate selectivity in several serine proteases. The impact of residue 190 on the selectivity was constant, regardless of differences in original selectivity or reactivity. Substrate binding in S1 was optimised in all wild-type enzymes, while the effects on k_cat depended on the combination of residue 190 and substrate. Mutagenesis of residue 190 did not affect the S2–S4 sites. Pronounced selectivity for arginine residues was coupled with low enzymatic activity, in particular in recombinant factor IXa. This is due to the dominance of the S1–P1 interaction over substrate binding in the S2–S4 sites.     

Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata

Three distinct alkaline serine proteases (named CTSP-1, -2, and -3) were purified from the polychaete Cirriformia tentaculata and characterized in terms of their enzymatic properties and kinetics. The estimated molecular masses of CTSP-1, -2, and -3 enzymes were found to be 28.8, 30.9, and 28.4 kDa, respectively. The enzymes were active at the temperature range of 50–60 °C under pH 8.5–9.0 and completely inactivated by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, but not by 1,10-phenanthroline and bestatin, suggesting that they are all typical serine proteases and not metalloproteases or cysteine proteases. CTSP-1 and -2 cleaved arginine, whereas CTSP-3 digested tyrosine residue at the carboxyl sides in their peptide substrates. A typical hepta-sequence (I-X-X-G-X-X-A) conserved in serine proteases from annelid species was found in N-termini of all CTSPs. CTSP-2 was the most active enzyme among the proteases purified as shown by kinetic values. The enzymes cleaved all chains of fibrinogen within 20 min and also hydrolyzed actively fibrin polymer as well as cross-linked fibrin. In addition, the enzymes could actively digest the fibrin clot in blood plasma milieu. Taken together, the results obtained demonstrate that CTSP enzymes have a potential of becoming therapeutic agents for thrombus dissolution.     

Elaborate manifold of short hydrogen bond arrays mediating binding of active site-directed serine protease inhibitors

An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog.Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.     

The 0.83 A resolution crystal structure of alpha-lytic protease reveals the detailed structure of the active site and identifies a source of conformational strain

The crystal structure of the extracellular bacterial serine protease α-lytic protease (αLP) has been solved at 0.83 Å resolution at pH 8. This ultra-high resolution structure allows accurate analysis of structural elements not possible with previous structures. Hydrogen atoms are visible, and confirm active-site hydrogen-bonding interactions expected for the apo enzyme. In particular, His57 N^δ1 participates in a normal hydrogen bond with Asp102 in the catalytic triad, with a hydrogen atom visible 0.83(±0.06) Å from the His N^δ1. The catalytic Ser195 occupies two conformations, one corresponding to a population of His57 that is doubly protonated, the other to the singly protonated His57. Based on the occupancy of these conformations, the pK_a of His57 is calculated to be ∼8.8 when a sulfate ion occupies the active site. This 0.83 Å structure has allowed critical analysis of geometric distortions within the structure. Interestingly, Phe228 is significantly distorted from planarity. The distortion of Phe228, buried in the core of the C-terminal domain, occurs at an estimated energetic cost of 4.1 kcal/mol. The conformational space for Phe228 is severely limited by the presence of Trp199, which prevents Phe228 from adopting the rotamer observed in many other chymotrypsin family members. In αLP, the only allowed rotamer leads to the deformation of Phe228 due to steric interactions with Thr181. We hypothesize that tight packing of co-evolved residues in this region, and the subsequent deformation of Phe228, contributes to the high cooperativity and large energetic barriers for folding and unfolding of αLP. The kinetic stability imparted by the large, cooperative unfolding barrier plays a critical role in extending the lifetime of the protease in its harsh environment.     

Molecular dynamics simulations investigate the pathway of substrate entry active site of rhomboid protease

Rhomboid proteases can catalyze peptide bond cleavage and participate in abundant biological processes encompassing all branches of life; however, the pathway for substrate entry into its active site remains ambiguous. Here, the two possible pathways are preliminarily determined through molecular dynamics: One pathway is between Tm2 and Tm5, and the other is between Loop3 and Loop5. Then, the umbrella sampling simulations are performed to investigate the more feasible pathway for substrate entry. The results show that free energy barriers along the two pathways are similar; in the pathway 1, Trp236 and Trp157 as pivotal residues are responsible for the rotation of substrate in the binding process; in the pathway 2, among some important residues, the residue His150 plays an important role in substrate entry. Further, combining with previous experiment results, it is concluded that the substrate is inclined to enter into the active site along pathway 2. Our results are important for further understanding the function and catalysis mechanism of rhomboid proteases.

Communicated by Ramaswamy H. Sarma     

Analysis of the context dependent sequence requirements of active site residues in the metallo-beta-lactamase IMP-1

The metallo-beta-lactamase IMP-1 catalyzes the hydrolysis of a broad range of beta-lactam antibiotics to provide bacterial resistance to these compounds. In this study, 29 amino acid residue positions in and near the active-site pocket of the IMP-1 enzyme were randomized individually by site-directed mutagenesis of the corresponding codons in the bla(IMP-1) gene. The 29 random libraries were used to identify positions that are critical for the catalytic and substrate-specific properties of the IMP-1 enzyme. Mutants from each of the random libraries were selected for the ability to confer to Escherichia coli resistance to ampicillin, cefotaxime, imipenem or cephaloridine. The DNA sequence of several functional mutants was determined for each of the substrates. Comparison of the sequences of mutants obtained from the different antibiotic selections indicates the sequence requirements for each position in the context of each substrate. The zinc-chelating residues in the active site were found to be essential for hydrolysis of all antibiotics tested. Several positions, however, displayed context-dependent sequence requirements, in that they were essential for one substrate(s) but not others. The most striking examples included Lys69, Asp84, Lys224, Pro225, Gly232, Asn233, Asp236 and Ser262. In addition, comparison of the results for all 29 positions indicates that hydrolysis of imipenem, cephaloridine and ampicillin has stringent sequence requirements, while the requirements for hydrolysis of cefotaxime are more relaxed. This suggests that more information is required to specify active-site pockets that carry out imipenem, cephaloridine or ampicillin hydrolysis than one that catalyzes cefotaxime hydrolysis.     

Crystal structure of earthworm fibrinolytic enzyme component a: revealing the structural determinants of its dual fibrinolytic activity

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida is a strong fibrinolytic enzyme that not only directly degrades fibrin, but also activates plasminogen. Proteolytic assays further revealed that it cleaved behind various P1 residue types. The crystal structure of EFEa was determined using the MIR method and refined to 2.3A resolution. The enzyme, showing the overall polypeptide fold of chymotrypsin-like serine proteases, possesses essential S1 specificity determinants characteristic of elastase. However, the beta strand at the west rim of the S1 specificity pocket is significantly elongated by a unique four-residue insertion (Ser-Ser-Gly-Leu) after Val217, which not only provides additional substrate hydrogen binding sites for distal P residues, but also causes extension of the S1 pocket at the south rim. The S2 subsite of the enzyme was partially occluded by the bulky side-chain of residue Tyr99. Structure-based inhibitor modeling demonstrated that EFEa's S1 specificity pocket was preferable for elastase-specific small hydrophobic P1 residues, while its accommodation of long and/or bulky P1 residues was also feasible if enhanced binding of the substrate and induced fit of the S1 pocket were achieved. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. The presence of Tyr99 at the S2 subsite indicates a preference for P2-Gly, while an induced fit of Tyr99 was also suggested for accommodation of bigger P2 residues. This structure is the first reported for an earthworm fibrinolytic enzyme component and serine protease originating from annelid worms.