Multiple forms of Bacillus subtilis subtilisin and effects of mutations on the distribution of its molecular forms]

Using polyacrylamide-gel electrophoresis, isoelectric focusing and gel-filtration it was demonstrated that the auxotrophic mutant strains of Bac. subtilis A-50 and their prototrophic revertant strains produce multiple molecular forms of subtilisin. Three of them are the same as the corresponding molecular forms of subtilisin from the wild strain A-50. In different mutant strains the relative amounts of the main three forms varies considerably resulting in the absence of certain forms in several strains. There is the additional minor form of subtilisin possessing high electrophoretic mobility in four prototrophic revertant strains and one Arg--auxotrophic strain of Bac. subtilis A-50. It would be reasonable to suppose that different molecular forms of subtilisin derive from the product of its single structural gene as a result of post-translational modifications (limited proteolysis). This enzyme and probably most, if not all secretory proteins may be synthesised as larger precursors and then specifically modified in the bacterial cell membranes. Thus, certain mutations, without affecting the structural gene of this secretory protein -- subtilisin -- have pronounced effects on this structural gene expression, varying the degree of its product modification and the amount of resulting secretory molecular forms of subtilisin.     

Effects of mutations in Bacillus subtilis genome decreasing the protease activity on the formation of subtilisin molecular forms]

Multiple molecular forms of subtilisin--extracellular serine protease produced by the wild strain Bac. subtilis A-50 and its mutant strains with the protease activity decreased two-fold and more were studied. Six molecular forms of subtilisin were found on the whole when 33 mutant strains have been investigated under the experimental conditions. It is essential that both the wild and each of mutant strains under study produced not more than three out of these six forms. Three molecular forms of subtilisin from the mutant strains are similar to those found in the wild strain A-50, and have the molecular weight, of 27 000-30 000. Three other forms of subtilisin were revealed only in the mutant strains, and had the molecular weight of about 20 000. Apparently there is only one structural gene for subtilisin in Bac. subtilis genome. The appearence of multiple molecular forms of subtilisin may be due to the post-translational modifications (limited proteolysis) of the initial type of enzyme, i.e. pre-subtilisin. Probably, that certain mulations not affecting the structural gene can significantly change the expression of such gene by varying of the degree of product modifications.     

Immunochemical study of subtilisins obtained from Bacillus subtilis A-50 and some of its mutants]

Physico-chemical parameters of subtilisins from the original Bacillus subtilis A-50 strain (proteolytic activity, electrophoretic mobility, molecular weight, reactions with specific inhibitors) were similar to those mentioned in the literature for the enzymes of other strains. Immunological experiment has shown, that Bacillus subtilis A-50 subtilisins with various electrophoretic mobility do not differ in their antigenic properties. Enzymes with high electrophoretic mobility from mutant strains were similar to I--III subtilisin fractions from Bac. subtilis A-50 in the antigenic characteristics. However, the antigenic heterogeneity was observed in I, II and III enzyme fractions of some mutant strains. Subtilisins studied appear to form the isoenzyme system.     

Biosynthesis of the antibiotic, grizin, by prototrophic revertants of Streptomyces griseus biochemical mutants

Spontaneous and nitrosomethylbiuret-induced prototrophic revertants of various biochemical mutants of Str. griseus producing grisin, a streptothricin antibiotic, were isolated. The antibiotic production level of the revertants was studied. It was found that most of the prototrophic revertants synthesized much higher amounts of grisin than the initial biochemical mutants. It was also shown that a number of the prototrophic revertants of the methionine- and arginine-dependent mutants synthesized 20-23% higher amounts of grisin as compared to the control.     

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.     

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.     

Affinity chromatography of subtilisin on a sorbent with epsilon-aminocapronyl-alanyl-alanyl-D-leucylamide. Detection of a serine protease with unusual properties]

A biospecific sorbent obtained by attachment of epsilon-aminocapronyl-L-alanyl-L-alanyl-D-leucylamide to CNBr-activated Sepharose 4B has been used for affinity chromatography of various samples of subtilisin BPN', e.g. subtilisin A ("Serva"), Nagarse, A-50. Two active components were isolated from subtilisin A (Serva"), the major component corresponding to subtilisin BPN' and the minor component (SII) being a serine proteinase with low molecular weight (about 10000). The molecular weight and amino acid composition of SII as well as the kinetic parameters of its action on peptide substrates (p-nitroanilides of N-benzyloxycarbonyl-Gly-Gly-Leu, -Ala-Ala-Leu, -Gly-Gly-Phe, -Ala-Ala-Phe. The low molecular weight proteinase possesses a high affinity for the leucine residue in P1 position and alanine in P2 and P3 positions. The specificity of this proteinase differs from that of the main component.     

Correlation between the virulence of streptococci for mice and their IgG FcR activity in in vivo experiments

The immunological analysis of 24 spontaneous Strr, Rifr and Kanr mutants of streptococcal strain IP, highly virulent for mice and capable of binding polyclonal human IgG (IgG FcR+), was made. The characteristic feature of all these mutants was decreased virulence, restored after their passage in vivo. 23 mutants were capable of binding polyclonal IgG; one Strr mutant had no such capacity, but acquired it, together with an increase in virulence, after its passage in vivo. When stored in meat-peptone agar without antibiotics, 5 out of 10 Strr mutants lost their capacity for binding polyclonal human IgG. After passage in vivo they regained this property simultaneously with virulence.     

Suppression by thymidine-requiring mutants of Escherichia coli K-12.

Thymidine-requiring strains of Escherichia coli isolated by trimethoprim selection often simultaneously acquire the ability to suppress bacteriophage T4 nonsense mutations. Suppression is lost in Thy+ revertants and recombinants, but is sometimes retained in thyA plasmid-bearing transformants. Suppression is restricted in Strr derivatives of the Thy- mutants, indicating that suppression occurs at the level of translation.     

Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression.

Concomitant activity improvement of an evolved enzyme toward two very different ester substrates was achieved when a unique combination of functional periplasmic enzyme expression in Escherichia coli, random mutagenesis, DNA shuffling and cell-based kinetic screenings was applied. Specifically, we focused on the conversion of subtilisin E into an enzyme with broader esterase activity as opposed to its native amidase activity. Cell-based microtiter assays were performed on N-acetyl-D,L-phenylalanine p-nitrophenyl ester (Phe-NPE) and sucrose 1'-adipate (S1'A), as well as on the tetrapeptide amide substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide. After a single modified cycle of directed molecular evolution, we isolated a number of clones exhibiting increased activity toward Phe-NPE. In the following rounds of screenings, mutants with improved activity on Phe-NPE were also tested on S1'A. Three mutants were identified with increased esterolytic activity on Phe-NPE and S1'A, while having similar amidase activity to that of the parental enzymes. Because the two ester substrates are structurally distinct, we have evolved a more general esterolytic subtilisin and this may have important applications in synthesis.     

Biochemical and immunological characterization of mutant L-M cells with altered levels of dibutyryl cyclic AMP-inducible alkaline phosphatase.

Phosphotyrosine-Sepharose 4B was synthesized and used to purify L-cell alkaline phosphatase. Antibodies to this enzyme interacted with the alkaline phosphatase of strains A-1-2 and A-3-3, mutants that express the enzyme constitutively. This and thermal stability studies suggest that these mutants contain the same alkaline phosphatase isozyme as their parent strain.     

Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis.     

Rapid folding of calcium-free subtilisin by a stabilized pro-domain mutant.

In vitro folding of mature subtilisin is extremely slow. The isolated pro-domain greatly accelerates in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In our studies of subtilisin, we are trying to answer two basic questions: why does subtilisin fold slowly without the pro-domain and what does the pro-domain do to accelerate the folding rate? To address these general questions, we are trying to characterize all the rate constants governing individual steps in the bimolecular folding reaction of pro-domain with subtilisin. Here, we report the results of a series of in vitro folding experiments using an engineered pro-domain mutant which is independently stable (proR9) and two calcium-free subtilisin mutants. The bimolecular folding reaction of subtilisin and proR9 occurs in two steps: an initial binding of proR9 to unfolded subtilisin, followed by isomerization of the initial complex into the native complex. The central findings are as follows. First, the independently stable proR9 folds subtilisin much faster than the predominantly unfolded wild-type pro-domain. Second, at micromolar concentrations of proR9, the subtilisin folding reaction becomes limited by the rate at which prolines in the unfolded state can isomerize to their native conformation. The simpliest mechanism which closely describes the data includes two denatured forms of subtilisin, which form the initial complex with proR9 at the same rate but which isomerize to the fully folded complex at much different rates. In this model, 77% of the subtilisin isomerizes to the native form slowly and the remaining 23% isomerizes more rapidly (1.5 s-1). The slow-folding population may be unfolded subtilisin with the trans form of proline 168, which must isomerize to the cis form during refolding. Third, in the absence of proline isomerization, the rate of subtilisin folding is rapid and at [proR9] 3 s-1. The implications of these results concerning why subtilisin folds slowly without the pro-domain are discussed.     

Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.     

Genetic and biochemical study of threonine-overproducing mutants of Saccharomyces cerevisiae.

Three threonine-overproducing mutants were obtained as prototrophic revertants of a hom3 mutant strain of Saccharomyces cerevisiae. The gene HOM3 codes for aspartokinase (aspartate kinase; EC 2.7.2.4), the first enzyme of the threonine-methionine biosynthetic route, which is subjected to feedback inhibition by threonine. Enzymatic studies indicated that aspartokinase from the revertants has lost the feedback inhibition, resulting in overproduction of threonine. These revertants also bore one or two additional mutations, named tex1-1 and tex2-1, which alone or jointly made possible the excretion of the threonine accumulated. The effect of these two genes on excretion is potentiated by excess inositol in the medium.     

Subtilisin of Leishmania amazonensis as Potential Druggable Target: Subcellular Localization,In Vitro Leishmanicidal Activity and Molecular Docking of PF-429242, a Subtilisin Inhibitor

Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug. In this study, we first show, by electron microscopy and flow cytometry, that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of humans were determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro. Here, PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC₅₀ values = 3.07 ± 0.20; 0.83 ± 0.12; 2.02 ± 0.27 and 5.83 ± 1.2 µM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC₅₀ = 170.30 µM). We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form; however, PF-429242 had a low effect against this intracellular form with an IC50 of >100 µM for intracellular amastigotes, as well as against axenic amastigotes (94.12 ± 2.8 µM for the LV78 strain). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets.     

Effect of nuclear mutation in maize on photosynthetic activity and content of chlorophyll-protein complexes

A number of new nuclear mutants have been isolated from maize by selection for high chlorophyll (Chl) fluorescence. These mutants show reduced rates of photosynthesis and/or are deficient in Chl. Electrophoretic examination of wild type thylakoid membranes revealed five Chl-protein complexes, two containing only Chl a and three containing Chl a and Chl b. A class of nonviable, photosystem I-deficient mutants was found to be lacking one (A-1) of the two Chl a-protein complexes. A second class of nonviable, photosystem I-lacking mutants was found to be missing not only this A-1 complex but also one or more of the three Chl a and b-containing, light-harvesting Chl-protein complexes. Viable mutants were obtained which appeared to have lost just one of the Chl b-containing complexes, whereas a second class of viable mutants was missing all three of the Chl b-complexes. The results confirm that the A-1 band is associated with the P700-Chl a-protein complex characterized previously. The data also indicate the existence of structurally different forms of the light-harvesting Chl a- and b-containing complexes. The results also show a lower molecular weight band (A-2) containing primarily Chl a and which appears to be required for viability.     

Pectinolytic activity of revertants of auxotrophic strains of Aspergillus niger

From conidia of 4 different auxotrophic A. niger strains 400 spontaneous revertants (100 from each strain) were obtained, and in one case additionally 100 revertants induced by mutagens (UV+NTG). The revertants showed a considerable differentiation with regard to the total pectinolytic activity. Its highest increase occurred in revertants originating from auxotrophs greatly predisposed to synthesize pectinases. In the case of revertants induced by mutagenes an increase in the frequency of their formation was observed, as well as an increased participation of revertants with higher pectinolytic activity compared to both their initial auxotrophic and prototrophic strain.     

Binding, proteolytic, and crystallographic analyses of mutations at the protease-inhibitor interface of the subtilisin BPN'/chymotrypsin inhibitor 2 complex

A series of mutants of chymotrypsin inhibitor 2 (CI2), at residues that interact with the inhibited enzyme subtilisin BPN', were studied to determine the relative importance of intermolecular contacts on either side of the scissile bond. Mutants were tested for inhibition of subtilisin, rates of hydrolysis by subtilisin, and ability to acylate subtilisin. Additionally, crystal structures of the mutant CI2 complexes with subtilisin were obtained. Ordered water molecules were found to play an important role in inhibitor recognition, and features of the crystal structures, in combination with biochemical data, support a transition-state stabilization role for the P(1) residue in subtilisin catalysis. Consistent with the proposed mechanism of inhibition, in which rapid acylation is followed by religation, leaving-group contacts with the enzyme were found to be more critical determinants of inhibition than acylating-group contacts in the mutants studied here.