Purification and Characterization of a Keratinolytic Serine Proteinase from Streptomyces albidoflavus

Streptomyces strain K_1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthetic substrates, and its remarkably high level of NH₂-terminal sequence homology with Streptomyces griseus protease B (SGPB) showed that the new enzyme, designated SAKase, was homologous to SGPB. We tested the activity of SAKase with soluble and fibrous substrates (elastin, keratin, and type I collagen) and found that it was very specific for keratinous substrates compared to SGPB and proteinase K.     

Heat stable proteinase from Thermomonospora fusca. Characterization as a serine proteinase

An extracellular proteinase secreted by the thermophilic bacteria Thermomonospora fusca YX (YX-proteinase) is a serine proteinase as shown by its inactivation by the site specific reagents, phenylmethanesulfonyl fluoride, dansyl fluoride, and carbobenzoxy-L-phenylalanine chloromethyl ketone. This conclusion is further supported by the effect of various proteinase inhibitors on its activity. The activity of the proteinase toward small synthetic ester substrates shows that the enzyme has a primary specificity for the aromatic and hydrophobic amino acids. The amino acid composition and NH2-terminal sequence, as well as its size, suggest that the enzyme is related to the chymotrypsin-like microbial proteinase, alpha-lytic protease from Myxobacter 495 and protease A and B from Streptomyces griseus.     

The 2.8 A resolution structure of Streptomyces griseus protease B and its homology with alpha-chymotrypsin and Streptomyces griseus protease A.

The 2.8 A (1 A = 0.1 nm) resolution structure of the crystalline orthorhombic form of the microbial serine protease Streptomyces griseus protease B (SGPB) has been solved by the method of multiple isomorphous replacement using five heavy-atom derivatives. The geometrical arrangement of the active site quartet, Ser-214, Asp-102, His-57, and Ser-195, is similar to that found for pancreatic alpha-chymotrypsin. SGPB and alpha-chymotrypsin have only 18% identity of primary structure but their tertiary structures are 63% topologically equivalent within a root mean square deviation of 2.07 A. The major tertiary structural differences between the bacterial enzyme SGPB and the pancreatic enzymes is due to the zymogen requirement of the multicellular organisms in order to protect themselves against autolytic degradation. The two pronase enzymes, SGPB and Streptomyces griseus protease A (SGPA), have 61% identity of sequence and their tertiary structures are 85% topologically equivalent within a root mean square deviation of 1.46 A. The active site regions of SGPA and SGPB are similar and their tertiary structures differ only in three minor regions of surface loops.     

The folding landscape of Streptomyces griseus protease B reveals the energetic costs and benefits associated with evolving kinetic stability

Like most extracellular bacterial proteases, Streptomyces griseus protease B (SGPB) and alpha-lytic protease (alphaLP) are synthesized with covalently attached pro regions necessary for their folding. In this article, we characterize the folding free energy landscape of SGPB and compare it to the folding landscapes of alphaLP and trypsin, a mammalian homolog that folds independently of its zymogen peptide. In contrast to the thermodynamically stable native state of trypsin, SGPB and alphaLP fold to native states that are thermodynamically marginally stable or unstable, respectively. Instead, their apparent stability arises kinetically, from unfolding free energy barriers that are both large and highly cooperative. The unique unfolding transitions of SGPB and alphaLP extend their functional lifetimes under highly degradatory conditions beyond that seen for trypsin; however, the penalty for evolving kinetic stability is remarkably large in that each factor of 2.4-8 in protease resistance is accompanied by a cost of ~10(5) in the spontaneous folding rate and ~5-9 kcal/mole in thermodynamic stability. These penalties have been overcome by the coevolution of increasingly effective pro regions to facilitate folding. Despite these costs, kinetic stability appears to be a potent mechanism for developing native-state properties that maximize protease longevity.     

Gene cloning and heterologous expression of a serine protease from Streptomyces fradiae var.k11

The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 degrees C for its caseinolytic activity and pH 9 and 62 degrees C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.     

Cleavage of peptide bonds bearing ionizable amino acids at P1 by serine proteases with hydrophobic S1 pocket

Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, and ∼10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S₁ pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.     

Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530.

A serine protease from the keratin-degrading Streptomyces pactum DSM 40530 was purified by casein agarose affinity chromatography. The enzyme had a molecular weight of 30,000 and an isoelectric point of 8.5. The proteinase was optimally active in the pH range from 7 to 10 and at temperatures from 40 to 75 degrees C. The enzyme was specific for arginine and lysine at the P1 site and for phenylalanine and arginine at the P1' site. It showed a high stereoselectivity and secondary specificity with different synthetic substrates. The keratinolytic activity of the purified proteinase was examined by incubation with the insoluble substrates keratin azure, feather meal, and native and autoclaved chicken feather downs. The S. pactum proteinase was significantly more active than the various commercially available proteinases. After incubation with the purified proteinase, a rapid disintegration of whole feathers was observed. But even after several days of incubation with repeated addition of enzymes, less than 10% of the native keratin substrate was solubilized. In the presence of dithiothreitol, degradation was more than 70%.     

Purification, characterization cloning, and sequencing of metalloendopeptidase from Streptomyces septatus TH-2

Streptomyces septatus TH-2 secretes a large amount of a protease when cultured on a medium containing K(2)HPO(4) and glucose. The enzyme was purified to homogeneity by a three-step procedure. This enzyme had a molecular mass of approximately 35kDa, and was particularly inhibited by EDTA and phosphoramidon. Its substrate specificity was investigated using novel fluorescence energy transfer combinatorial libraries. The protease was found to prefer Phe and Tyr at the P(1) position, a hydrophobic or basic residue at the P(2) position, and a basic or small residue at the P(3) position. Its gene was cloned and sequenced, and its deduced amino acid sequence contained an HEXXH consensus sequence for zinc binding, confirming that it encodes metalloendopeptidase. The primary structure of the enzyme showed 40 and 69% identities with that of thermolysin from Bacillus thermoproteolyticus and that of a metalloendopeptidase from Streptomyces griseus, respectively.     

Contribution of peptide bonds to inhibitor-protease binding: crystal structures of the turkey ovomucoid third domain backbone variants OMTKY3-Pro18I and OMTKY3-psi[COO]-Leu18I in complex with Streptomyces griseus proteinase B (SGPB) and the structure of the free inhibitor, OMTKY-3-psi[CH2NH2+]-Asp19I

X-ray crystallography has been used to determine the 3D structures of two complexes between Streptomyces griseus proteinase B (SGPB), a bacterial serine proteinase, and backbone variants of turkey ovomucoid third domain (OMTKY3). The natural P1 residue (Leu18I) has been substituted by a proline residue (OMTKY3-Pro18I) and in the second variant, the peptide bond between Thr17I and Leu18I was replaced by an ester bond (OMTKY3-psi[COO]-Leu18I). Both variants lack the P1 NH group that donates a bifurcated hydrogen bond to the carbonyl O of Ser214 and O(gamma) of the catalytic Ser195, one of the common interactions between serine proteinases and their canonical inhibitors. The SGPB:OMTKY3-Pro18I complex has many structural differences in the vicinity of the S1 pocket when compared with the previously determined structure of SGPB:OMTKY3-Leu18I. The result is a huge difference in the DeltaG degrees of binding (8.3 kcal/mol), only part of which can be attributed to the missing hydrogen bond. In contrast, very little structural difference exists between the complexes of SGPB:OMTKY3-psi[COO]-Leu18I and SGPB:OMTKY3-Leu18I, aside from an ester O replacing the P1 NH group. Therefore, the difference in DeltaG degrees, 1.5 kcal/mol as calculated from the measured equilibrium association constants, can be attributed to the contribution of the P1 NH hydrogen bond toward binding. A crystal structure of OMTKY3 having a reduced peptide bond between P1 Leu18I and P'1 Asp19I, (OMTKY3-psi[CH2NH2+]-Asp19I) has also been determined by X-ray crystallography. This variant has very weak association equilibrium constants with SGPB and with chymotrypsin. The structure of the free inhibitor suggests that the reduced peptide bond has not introduced any major structural changes in the inhibitor. Therefore, its poor ability to inhibit serine proteinases is likely due to the disruptions of the canonical interactions at the oxyanion hole.     

Alkaline serine protease produced by Streptomyces sp. degrades PrP(Sc)

A PrP(Sc)-degrading enzyme was isolated from the culture medium of Streptomyces sp. using perchloric acid-soluble protein (PSP) as a substrate. The media of 500 microbial species were screened to obtain the PSP-degrading enzyme. The medium containing the protease secreted from strain 99-GP-2D-5 showed the highest PSP-degrading activity. Strain 99-GP-2D-5 was assigned as the genus Streptomyces by its morphological and chemotaxonomic characteristics. When scrapie prion was used as the substrate, it was completely digested by the enzyme. The amino acid sequence of the enzyme was identical to that of the C-terminal region of alkaline serine protease (ASP) I. ASP I may be the precursor of the enzyme, and the enzyme seems to be the mature type of ASP I. The maximal activity of the enzyme was observed at 60 degrees C and pH 11, and the scrapie prion was degraded within 3 min under the optimum conditions.     

Interactions of Streptomyces subtilisin inhibitor with Streptomyces griseus proteases A and B. Enzyme kinetic and computer simulation studies

Streptomyces subtilisin inhibitor (SSI), a dimeric protein that strongly inhibits subtilisins, was shown to form tight inhibitory complexes with Streptomyces griseus proteases A and B (SGPA and SGPB). The apparent dissociation constants of the SGPA-SSI and SGPB-SSI complexes were found to be orders of magnitude less than those of subtilisin-SSI complexes. Using the known atomic coordinates for SGPA and SSI, the highly complementary nature of the surface geometries of the two proteins was confirmed by a computer graphics study, which led to a proposed structure for the SGPA-SSI complex. Kinetic studies further suggested that the SSI dimer can bind two molecules of either SGPA or SGPB, and the 2:1-complexes (consisting of one inhibitor dimer and one enzyme molecule) apparently possess lower intrinsic dissociation constants than the 2:2-complexes. It was also shown that both of SGPA and SGPB are inhibited by both soybean trypsin inhibitor (Kunitz) and bovine pancreatic trypsin inhibitor (Kunitz), but far less strongly than by SSI.     

Maleylation and pH-dependence of Streptomyces griseus protease B.

The enzymatic activity of Streptomyces griseus protease B (SGPB) was measured over pH range 8.4--11.5 using a specific new, chromophoric substrate N-succinyl-glycyl-glycyl-L-phenylalanine p-nitroanilide. It was found that the activity is dependent on ionization of a single group with apparent pK = 10.84, possibly lysine-125. Maleylation of the epsilon-amino group of this lysine was linearily associated with the loss of enzymatic activity. It is therefore suggested that the electrostatic interaction between the side chain of lysine-125 and the alpha-carboxyl group of the C-terminal tyrosine is crucial to the active conformation of the enzyme. In contrast the maleylation of the alpha-amino group of the N-terminal isoleucine was rapid but could not be correlated to the loss of activity.     

Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-tag

In order to produce recombinant microbial transglutaminase (rMTG) which is free of the activating protease, dispase was used to activate the pro-rMTG followed by immobilized metal affinity chromatography (IMAC). As shown by MALDI-MS, the dispase does not only cleave the pro-sequence, but unfortunately also cleaves within the C-terminal histidine-tag. Hence, the active rMTG cannot properly bind to the IMAC material. As an alternative, proteinase K was investigated. This protease was successfully applied for the activation of purified pro-rMTG either as free or immobilized enzyme and the free enzyme was also applicable directly in the crude cell extract of E. coli. Thus, it enables a simple two-step activation/purification procedure resulting in protease-free and almost pure transglutaminase preparations. The protocol has been successfully applied to both, wild-type transglutaminase of Streptomyces mobaraensis as well as to the highly active variant S2P. Proteinase K activates the pro-rMTG without unwanted degradation of the histidine-tag. It turned out to be very important to inhibit proteinase K activity, e.g., by PMSF, prior to protein separation by SDS-PAGE.     

Water molecules participate in proteinase-inhibitor interactions: crystal structures of Leu18, Ala18, and Gly18 variants of turkey ovomucoid inhibitor third domain complexed with Streptomyces griseus proteinase B.

Crystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala18, and Gly18, have been determined and refined to high resolution. Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S1 binding pocket. The number and relative positions of water molecules bound in the S1 binding pocket vary according to the size of the side chain of the P1 residue. Water molecules in the S1 binding pocket of SGPB are redistributed in response to the complex formation, probably to optimize hydrogen bonds between the enzyme and the inhibitor. There are extensive water-mediated hydrogen bonds in the interfaces of the complexes. In all complexes, Asn 36 of OMTKY3 participates in forming hydrogen bonds, via water molecules, with residues lining the S1 binding pocket of SGPB. For a homologous series of aliphatic straight side chains, Gly18, Ala18, Abu18, Ape18, and Ahp18 variants, the binding free energy is a linear function of the hydrophobic surface area buried in the interface of the corresponding complexes. The resulting constant of proportionality is 34.1 cal mol-1 A-2. These structures confirm that the binding of OMTKY3 to the preformed S1 pocket in SGPB involves no substantial structural disturbances that commonly occur in the site-directed mutagenesis studies of interior residues in other proteins, thus providing one of the most reliable assessments of the contribution of the hydrophobic effect to protein-complex stability.     

Generation of a thermostable and denaturant-resistant peptide ligase

The construction and characterization of a novel, thermostable, peptide ligase are described. Three amino acid substitutions were introduced into the secreted bacterial protease Streptomyces griseus protease B (SGPB). Mutations were chosen on the basis of two separate observations: (i) that a single substitution of the nucleophilic serine (S195A) created an enzyme with significant peptide-ligation activity, albeit greatly reduced stability [(2000) Chem. Biol. 7, 163], and (ii) that a pair of substitutions in the substrate-binding pocket (T213L and F228H) greatly increased the thermostability of the wild-type enzyme [(1996) J. Mol. Biol. 257, 233]. The triple mutant, named streptoligase, was found to catalyze peptide ligation (aminolysis of both a thiobenzyl ester and a p-nitroanilide-activated peptide) efficiently in nondenaturing and denaturing conditions including SDS (0.5% w/v) and guanidine hydrochloride (4.0 M). Moreover, streptoligase exhibited a half-live for unfolding of 16.3 min at 55 degrees C in the absence of stabilizing substrates. The fraction of the streptoligase-catalyzed reaction that gave coupled product with the acceptor peptide FAASR-NH(2) was greater for the p-nitroanilide donor (Sc-AAPF-pNA) than for the benzyl thioester substrate (Sc-AAPF-SBn). These observations are consistent with ligation proceeding through an acyl-enzyme intermediate involving histidine-57. In the case of the thioester donor the triple mutant promotes the direct attack of water on the thioester carbonyl carbon, in addition to hydrolysis occurring at the stage of the acyl-enzyme intermediate. The strategy of multiple point mutations outlined in this study may provide a general means of converting enzymes with chymotrypsin-like protein folds into peptide ligases.     

Functional expression of the keratinolytic serine protease gene sfp2 from Streptomyces fradiae var. k11 in Pichia pastoris

We report the initial characterization and expression of sfp2, a gene encoding a keratinolytic serine protease from Streptomyces fradiae var. k11. Recombinant SFP2 was expressed in and secreted from the yeast Pichia pastoris with a final yield of 78 mg/L (136.2 U/mL caseinolytic activity) after 25 h of induction. The recombinant enzyme was purified using by ammonium sulfate precipitation and gel filtration chromatography to electrophoretic homogeneity, which was appropriately glycosylated and had a molecular mass of 26.0 kDa. The purified recombinant SFP2 was characterized. The optimal pHs and temperatures of SFP2 for proteolysis of casein and keratin azure were pH 10.0, 60 degrees C, and pH 9.0, 55 degrees C, respectively. SFP2 activity was stable from pH 3.0 to pH 11.0. The enzyme activity was inhibited by Co(2+) and Cr(3+) and enhanced by Ni(2+) and Cu(2+). The K(m) of 0.45 mmol/L and V(max) of 19.84 mmol/min mg were calculated using N-succinyl-Ala-Ala-Pro-Phe-pNA as a substrate. We tested the activity of SFP2 with soluble and insoluble substrates; SFP2 was more specific for keratinous substrates compared with proteinase K and other commercial proteases.     

Comprehensive analysis of protein folding activation thermodynamics reveals a universal behavior violated by kinetically stable proteases

Alpha-lytic protease (alpha LP) and Streptomyces griseus protease B (SGPB) are two extracellular serine proteases whose folding is absolutely dependent on the existence of their companion pro regions. Moreover, the native states of these proteins are, at best, marginally stable, with the apparent stability resulting from being kinetically trapped in the native state by large barriers to unfolding. Here, in an effort to understand the physical properties that distinguish kinetically and thermodynamically stable proteins, we study the temperature-dependences of the folding and unfolding kinetics of alpha LP and SGPB without their pro regions, and compare their behavior to a comprehensive set of other proteins. For the folding activation thermodynamics, we find some remarkable universal behaviors in the thermodynamically stable proteins that are violated dramatically by alpha LP. Despite significant variations in deltaC(P,F)++, the maximal folding speed occurs within the narrow biological temperature range for all proteins, except for alpha LP, with its maximal folding speed shifted lower by 200 K. This implies evolutionary pressures on folding speed for typical proteins, but not for alpha LP. In addition, the folding free energy barrier in the biological temperature range for most proteins is predominantly enthalpic, but purely entropic for alpha LP. The unfolding of alpha LP and SGPB is distinguished by three properties: a remarkably large deltaC(P,U)++, a very high deltaG(U)++, and a maximum deltaG(u)++ at the optimal growth temperature for the organism. While other proteins display each of these traits to some approximation, the simultaneous optimization of all three occurs only in the kinetically stable proteins, and appears to be required to maximize their unfolding cooperativity, by suppressing local unfolding events, and slowing the rate of global unfolding. Together, these properties extend the lifetime of these enzymes in the highly proteolytic extracellular environment. Attaining such functional properties seems possible only through the gross perturbation of the folding thermodynamics, which in turn has required the co-evolution of pro regions as folding catalysts.     

Purification and characterization of a multicatalytic proteinase from Atlantic salmon (Salmo salar) muscle

A high molecular mass alkaline proteinase was purified by DEAE-Sepharose and Mono Q chromatography. The mol. wt was estimated to be about 600,000. Under denaturing conditions, the enzyme dissociated into a cluster of subunits with mol. wt ranging from 25,000 to 30,000. The isoelectric point of the enzyme was about pH 7.3. The proteinase was able to hydrolyse N-terminal-blocked 4-methyl-7-coumarylamide substrates for either trypsin- or chymotrypsin-like activity. It was also able to hydrolyse haemoglobin and myosin at temperatures of about 60°C. The activities responded to pH and some chemicals in different ways. The trypsin-like activity was clearly inhibited by several serine protease inhibitors. These results suggest that the enzyme is multicatalytic, having at least two different active sites.     

Deleterious effects of beta-branched residues in the S1 specificity pocket of Streptomyces griseus proteinase B (SGPB): crystal structures of the turkey ovomucoid third domain variants Ile18I, Val18I, Thr18I, and Ser18I in complex with SGPB

Turkey ovomucoid third domain (OMTKY3) is a canonical inhibitor of serine proteinases. Upon complex formation, the inhibitors fully exposed P1 residue becomes fully buried in the preformed cavity of the enzyme. All 20 P1 variants of OMTKY3 have been obtained by recombinant DNA technology and their equilibrium association constants have been measured with six serine proteinases. To rationalize the trends observed in this data set, high resolution crystal structures have been determined for OMTKY3 P1 variants in complex with the bacterial serine proteinase, Streptomyces griseus proteinase B (SGPB). Four high resolution complex structures are being reported in this paper; the three beta-branched variants, Ile18I, Val18I, and Thr18I, determined to 2.1, 1.6, and 1.7 A resolution, respectively, and the structure of the Ser18I variant complex, determined to 1.9 A resolution. Models of the Cys18I, Hse18I, and Ape18I variant complexes are also discussed. The beta-branched side chains are not complementary to the shape of the S1 binding pocket in SGPB, in contrast to that of the wild-type gamma-branched P1 residue for OMTKY3, Leu18I. Chi1 angles of approximately 40 degrees are imposed on the side chains of Ile18I, Val18I, and Thr18I within the S1 pocket. Dihedral angles of +60 degrees, -60 degrees, or 180 degrees are more commonly observed but 40 degrees is not unfavorable for the beta-branched side chains. Thr18I Ogamma1 also forms a hydrogen bond with Ser195 Ogamma in this orientation. The Ser18I side chain adopts two alternate conformations within the S1 pocket of SGPB, suggesting that the side chain is not stable in either conformation.     

Hydrolysis of native proteins by keratinolytic protease of Doratomyces microsporus

A keratinolytic protease from the fungus Doratomyces microsporus was investigated for its ability to hydrolyse different native proteins. The purified enzyme was incubated for up to 24 h with keratinous substrates as well as with non-keratinous proteins. The results showed that the enzyme was broad specific since it hydrolysed various globular and fibrillar proteins. The hydrolysis of keratinous substrates decreased in the following order: skin keratins > nail keratins > hair keratins. With non-keratinous substrates, the order was: casein > BSA > elastin. Feather keratin and collagen could not be hydrolysed. Comparison of the enzyme with some known proteolytic enzymes showed that on keratin from stratum corneum the activity of the keratinase was comparable to that of proteinase K, other enzymes were less active. Hydrolysis of porcine skin with the keratinase revealed the degradation of the epidermis while dermis was not damaged.