Binase-Induced Changes of Tumor Cell Membranes

Exogenous ribonucleases of Bacilli can selectively induce apoptosis of malignant cells. The ability of Bacillus pumilus ribonuclease, binase, to induce processes leading to a dynamic disruption of the integrity of A549 human pulmonary adenocarcinoma cell membranes was analyzed. The influence of different enzyme concentrations on the state of the cytoplasmic membrane of cells and mitochondrial membranes was characterized. Using the methods of flow cytofluorometry and fluorescence microscopy, it has been established that binase leads to disruption in normal functioning of both types of membranes, with mitochondrial membranes affected first. The study made it possible to identify and visualize the effects of binase on the membrane structures of target cells and to confirm that bacterial RNase induces apoptosis of target cells mainly through the “internal” (mitochondrial) pathway.     

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.     

Brownian dynamics simulation of the competitive reactions: binase dimerization and the association of binase and barstar

A comparative study of the competitive reactions-the association reaction of binase with polypeptide inhibitor barstar and the reaction of binase dimerization-has been performed by the Brownian dynamics simulation method. It was shown that three types of the binase dimers could be formed and the dimerization reaction could compete with the inhibition reaction. The first type of the dimers leaves the active centre of binase free. During the formation of the dimers of the second and the third types the active centre of one or both binase molecules is blocked and ribonuclease becomes partially or fully inactive. Brownian dynamics simulation shown, that the ratio of competitive reaction rates depends on pH and ionic strength of solution.     

Homology modeling of the insect chitinase catalytic domain--oligosaccharide complex and the role of a putative active site tryptophan in catalysis

Knowledge-based protein modeling and substrate docking experiments as well as structural and sequence comparisons were performed to identify potential active-site residues in chitinase, a molting enzyme from the tobacco hornworm, Munduca sexta. We report here the identification of an active-site amino acid residue, W145. Several mutated forms of the gene encoding this protein were generated by site-directed mutagenesis, expressed in a baculovirus-insect cell-line system, and the corresponding mutant proteins were purified and characterized for their catalytic and substrate-binding properties. W145, which is present in the presumptive catalytic site, was selected for mutation to phenylalanine (F) and glycine (G), and the resulting mutant enzymes were characterized to evaluate the mechanistic role of this residue. The wild-type and W145F mutant proteins exhibited similar hydrolytic activities towards a tri-GlcNAc oligosaccharide substrate, but the former was approximately twofold more active towards a polymeric chitin-modified substrate. The W145G mutant protein was inactive towards both substrates, although it still retained its ability to bind chitin. Therefore, W145 is required for optimal catalytic activity but is not essential for binding to chitin. Measurement of kinetic constants of the wild-type and mutant proteins suggests that W145 increases the affinity of the enzyme for the polymeric substrate and also extends the alkaline pH range in which the enzyme is active.     

The crystal structure of an esterase from the hyperthermophilic microorganism <Emphasis Type="Italic">Pyrobaculum calidifontis</Emphasis> VA1 explains its enantioselectivity

The highly thermostable esterase from the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 (PestE) shows high enantioselectivity (E?>?100) in the kinetic resolution of racemic chiral carboxylic acids, but little selectivity towards acetates of tertiary alcohols (E?=?2–4). To explain these unique properties, its crystal structure has been determined at 2.0 Å resolution. The enzyme is a member of the hormone-sensitive lipase group (group H) of the esterase/lipase superfamily on the basis of the amino acid sequence identity. The PestE structure shows a canonical α/β-hydrolase fold as core domain with a cap structure at the C-terminal end of the β-sheet. A tetramer in the crystal packing is formed of two dimers; the dimeric form is observed in solution. Conserved dimers and even tetramers are found in other group H proteins. The amino acid residues Ser157, His284, and Asp254 form the catalytic triad, which is typically found in α/β-hydrolases. The oxyanion hole is composed of Gly85 and Gly86 within the conserved sequence motif HGGG(M,F,W) (amino acid residues 83–87) and Ala158. With the elucidated structure, experimental results about enantioselectivity towards the two model substrate classes (as exemplified for 3-phenylbutanoic acid ethyl ester and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate) could be explained by molecular modeling. For both enantiomers of the tertiary alcohol, orientations in two binding pockets were obtained without significant energy differences corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the carboxylic acid ester, two different orientations with significant energy differences for each enantiomer were found matching the high E values.     

Structural basis for the substrate specificity of 3-hydroxybutyrate dehydrogenase

The substrate specificity of 3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis with a non-native substrate, levulinic acid, was studied by analysis of the enzyme-substrate molecular interactions. The relation between structural and kinetic parameters was investigated considering the catalytic mechanism of the enzyme. The effects of key positive mutations (H144L, H144L/W187F) on the catalytic activity of the enzyme were studied by employing a surface analysis of its interatomic contacts between the enzyme and substrate atoms. The results revealed that the alteration of hydrogen bond network and rearrangement of the hydrophobic interactions between the active site and substrate molecule are the key structural basis for the change of the substrate specificity of 3-hydroxybutyrate dehydrogenase toward levulinic acid. With this approach, the structural basis for the substrate specificity of the enzyme could be elucidated in a quantitative manner.     

Crystal structures of a low-molecular weight protein tyrosine phosphatase from Saccharomyces cerevisiae and its complex with the substrate p-nitrophenyl phosphate

Low-molecular weight protein tyrosine phosphatases are virtually ubiquitous, which implies that they have important cellular functions. We present here the 2.2 A resolution X-ray crystallographic structure of wild-type LTP1, a low-molecular weight protein tyrosine phosphatase from Saccharomyces cerevisiae. We also present the structure of an inactive mutant substrate complex of LTP1 with p-nitrophenyl phosphate (pNPP) at a resolution of 1.7 A. The crystal structures of the wild-type protein and of the inactive mutant both have two molecules per asymmetric unit. The wild-type protein crystal was grown in HEPES buffer, a sulfonate anion that resembles the phosphate substrate, and a HEPES molecule was found with nearly full occupancy in the active site. Although the fold of LTP1 resembles that of its bovine counterpart BPTP, there are significant changes around the active site that explain differences in their kinetic behavior. In the crystal of the inactive mutant of LTP1, one molecule has a pNPP in the active site, while the other has a phosphate ion. The aromatic residues lining the walls of the active site cavity exhibit large relative movements between the two molecules. The phosphate groups present in the structures of the mutant protein bind more deeply in the active site (that is, closer to the position of nucleophilic cysteine side chain) than does the sulfonate group of the HEPES molecule in the wild-type structure. This further confirms the important role of the phosphate-binding loop in stabilizing the deep binding position of the phosphate group, thus helping to bring the phosphate close to the thiolate anion of nucleophilic cysteine, and facilitating the formation of the phosphoenzyme intermediate.     

Exogenous <Emphasis Type="Italic">Bacillus pumilus</Emphasis> RNase (binase) suppresses the reproduction of reovirus serotype 1

The experimental study identified the antiviral activity of Bacillus pumilus RNase (binase) against the reovirus of serotype 1/strain Lang. For the first time, it has been found that 50 μg/mL of binase effectively reduced the hemagglutinin and cytocidal activity of reovirus in Vero cell line. The preincubation of the enzyme with reovirus before infection of the cells inhibited the viral replication. To determine the stagedependent effect of reovirus reproduction upon binase inhibition, the infected cells were treated with binase or RNase A at different phases of the infectious cycle. The treatment of virus-infected cells has revealed that both enzymes have a maximal antiviral effect on the reovirus propagation during early phases of the reovirus reproduction cycle, with binase being more effective than RNase A. It has been hypothesized that the combined action of the oncolytic reovirus and binase is promising for the elimination of tumor cells carrying mutated RAS gene.     

Conformation of 2'GMP bound to a mutant ribonuclease T1 (Y45W) determined by X-ray diffraction and NMR methods.

The crystal structure of a mutant ribonuclease T1 (Y45W) complexed with a specific inhibitor, 2'GMP, has been determined by X-ray diffraction and refined at 1.9 A resolution to a conventional R-factor of 0.164. The mode of recognition of the guanine base by the enzyme is similar to that found for the wild-type ribonuclease T1 complexed with 2'GMP. The binding of the guanine base is clearly enhanced by maximum overlapping of the indole ring of Trp45 and the base. The glycosyl torsion angle of the inhibitor is in the syn conformation and the sugar exhibits a C3'-endo type pucker, which differs from that observed in the crystal of the complex between the wild-type ribonuclease T1 and 2'GMP. Analysis of 500-MHZ NMR spectra has also indicated that the 2'GMP molecule as bound to the mutant enzyme in solution exhibits a C3'-endo type pucker, similar to that bound to the wild-type enzyme in solution [Inagaki, Shimada, & Miyazawa (1985) Biochemistry 24, 1013-1020].     

Stabilization of a reaction intermediate as a catalytic device: definition of the functional role of the flexible loop in triosephosphate isomerase

The function of the mobile loop of triosephosphate isomerase has been investigated by deleting four contiguous residues from the part of this loop that interacts directly with the bound substrate. From the crystal structure of the wild-type enzyme, it appears that this excision will not significantly alter the conformation of the rest of the main chain of the protein. The specific catalytic activity of the purified mutant enzyme is nearly 10(5)-fold lower than that of the wild type. Kinetic measurements and isotopic partitioning studies show that the decrease in activity is due to much higher activation barriers for the enolization of enzyme-bound substrate. Although the substrates bind somewhat more weakly to the mutant enzyme than to the wild type, the intermediate analogue phosphoglycolohydroxamate binds much less well (by 200-fold) to the mutant. It seems that the deleted residues of the loop contribute critically to the stabilization of the enediol phosphate intermediate. Consistent with this view, the mutant enzyme can no longer prevent the loss of the enediol phosphate from the active site and its rapid decomposition to methylglyoxal and inorganic phosphate. Indeed, when glyceraldehyde 3-phosphate is the substrate, the enediol phosphate intermediate is lost (and decomposes) 5.5 times faster than it reprotonates to form the product dihydroxyacetone phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steady-state kinetic analysis of phosphotransacetylase from Methanosarcina thermophila

Phosphotransacetylase (EC 2.3.1.8) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. A steady-state kinetic analysis of the phosphotransacetylase from Methanosarcina thermophila indicated that there is a ternary complex kinetic mechanism rather than a ping-pong kinetic mechanism. Additionally, inhibition patterns of products and a nonreactive substrate analog suggested that the substrates bind to the enzyme in a random order. Dynamic light scattering revealed that the enzyme is dimeric in solution.     

Catalytically important amino acid residues in endoglucanases from a mutant strain <Emphasis Type="Italic">Trichoderma</Emphasis> sp. M<Subscript>7</Subscript>

Two endoglucanases, EG-III (49.7 kD) and EG-IV (47.5 kD), from a mutant strain Trichoderma sp. M₇ were modified with several specific reagents. Water-soluble carbodiimide completely inactivated only one of the purified endoglucanases and kinetic analysis indicated that at least two molecules of carbodiimide bind to EG-IV for inactivation. The reaction followed pseudo-first-order kinetics with a second-order rate constant of 3.57·10^?5 mM^?1·min^?1. Both endoglucanases were inhibited by iodoacetamide, but the absence of substrate protection excluded direct involvement of cysteine residues in the catalysis N-Bromosuccinimide (NBS) showed a strong inhibitory effect on both endoglucanases, suggesting that tryptophan residues are essential for the activity and binding to the substrate, since the presence of substrates or analogs prior to NBS modification protected the enzymes against inactivation.     

Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

An important active-site residue in the glycolytic enzyme triosephosphate isomerase is His-95, which appears to act as an electrophilic component in catalyzing the enolization of the substrates. With the techniques of site-directed mutagenesis, His-95 has been replaced by Gln in the isomerase from Saccharomyces cerevisiae. The mutant isomerase has been expressed in Escherichia coli strain DF502 and purified to homogeneity. The specific catalytic activity of the mutant enzyme is less than that of wild type by a factor of nearly 400. The mutant enzyme can be resolved from the wild-type isomerase on nondenaturing isoelectric focusing gels, and an isomerase activity stain shows that the observed catalytic activity indeed derives from the mutant protein. The inhibition constants for arsenate and for glycerol phosphate with the mutant enzyme are similar to those with the wild-type isomerase, but the substrate analogues 2-phosphoglycolate and phosphoglycolohydroxamate bind 8- and 35-fold, respectively, more weakly to the mutant isomerase. The mutant enzyme shows the same stereospecificity of proton transfer as the wild type. Tritium exchange experiments similar to those used to define the free energy profile for the wild-type yeast isomerase, together with a new method of analysis involving 14C and 3H doubly labeled substrates, have been used to investigate the energetics of the mutant enzyme catalyzed reaction. When the enzymatic reaction is conducted in tritiated solvent, the mutant isomerase does not catalyze any appreciable exchange between protons of the remaining substrate and those of the solvent either in the forward reaction direction (using dihydroxyacetone phosphate as substrate) or in the reverse direction (using glyceraldehyde phosphate as substrate). However, the specific radioactivity of the product glyceraldehyde phosphate formed in the forward reaction is 31% that of the solvent, while that of the product dihydroxyacetone phosphate formed in the reverse reaction is 24% that of the solvent. The deuterium kinetic isotope effects observed with the mutant isomerase using [1(R)-2H]dihydroxyacetone phosphate and [2-2H]glyceraldehyde 3-phosphate are 2.15 +/- 0.04 and 2.4 +/- 0.1, respectively. These results lead to the conclusion that substitution of Gln for His-95 so impairs the ability of the enzyme to stabilize the reaction intermediate that there is a change in the pathways of proton transfer mediated by the mutant enzyme. The data allow us more closely to define the role of His-95 in the reaction catalyzed by the wild-type enzyme, while forcing us to be alert to subtle changes in mechanistic pathways when mutant enzymes are generated.     

Inorganic phosphate blocks binding of pre‐miRNA to Dicer‐2 via its PAZ domain

In Drosophila, Dicer‐1 produces microRNAs (miRNAs) from pre‐miRNAs, whereas Dicer‐2 generates small interfering RNAs from long double‐stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer‐2 cleavage of pre‐miRNAs, but not long dsRNAs. Here, we report that phosphate‐dependent substrate discrimination by Dicer‐2 reflects dsRNA substrate length. Efficient processing by Dicer‐2 of short dsRNA requires a 5′ terminal phosphate and a two‐nucleotide, 3′ overhang, but does not require ATP. Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer dsRNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer‐2 PAZ domain blocked cleavage of short, but not long, dsRNA. We propose that inorganic phosphate occupies a PAZ domain pocket required to bind the 5′ terminal phosphate of short substrates, blocking their use and restricting pre‐miRNA processing in flies to Dicer‐1. Our study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme.     

A novel secreted ribonuclease from Bacillus intermedius: gene structure and regulatory control

A second secreted ribonuclease, designated binase II, has been detected in Bacillus intermedius 7P, and its structural gene was cloned and sequenced. Unlike the well-known binase I, a 109-amino acid guanyl-specific enzyme, the 292-residue binase II is closely related to the B. subtilis nuclease Bsn, in structure and in its enzymatic properties. Binase II is also insensitive to inactivation by barstar, an inhibitor protein that is specific for guanyl-specific ribonucleases. While both B. intermedius enzymes are induced upon phosphate starvation, only the gene for binase I belongs to the pho regulon system and carries pho-box elements adjacent to its promoter sequence. The gene for binase II is similar to that for Bsn in lacking such elements. The birB gene coding for binase II appears to be located next to the 3′-end of a ferric ion transport operon, with which it convergently overlaps. This would allow attenuator control over binase II expression under conditions of starvation for ferric ions. Received: 12 October 1999 / Accepted: 10 February 2000     

Catalysis by Escherichia coli ribonuclease HI is facilitated by a phosphate group of the substrate

To investigate the role of the phosphate group 3' to the scissile phosphodiester bond of the substrate in the catalytic mechanism of Escherichia coli ribonuclease HI (RNase HI), we have used modified RNA-DNA hybrid substrates carrying a phosphorothioate substitution at this position or lacking this phosphate group for the cleavage reaction. Kinetic parameters of the H124A mutant enzyme, in which His(124) was substituted with Ala, as well as those of the wild-type RNase HI, were determined. Substitution of the pro-R(p)-oxygen of the phosphate group 3' to the scissile phosphodiester bond of the substrate with sulfur reduced the k(cat) value of the wild-type RNase HI by 6.9-fold and that of the H124A mutant enzyme by only 1. 9-fold. In contrast, substitution of the pro-S(p)-oxygen of the phosphate group at this position with sulfur had little effect on the k(cat) value of the wild-type and H124A mutant enzymes. The results obtained for the substrate lacking this phosphate group were consistent with those obtained for the substrates with the phosphorothioate substitutions. In addition, a severalfold increase in the K(m) value was observed by the substitution of the pro-R(p)-oxygen of the substrate with sulfur or by the substitution of His(124) of the enzyme with Ala, suggesting that a hydrogen bond is formed between the pro-R(p)-oxygen and His(124). These results allow us to propose that the pro-R(p)-oxygen contributes to orient His(124) to the best position for the catalytic function through the formation of a hydrogen bond.     

Aspartic-129 is an essential residue in the catalytic mechanism of the low Mr phosphotyrosine protein phosphatase

The crystal structure of the bovine liver low M_r phosphotyrosine protein phosphatase suggests the involvement of aspartic acid-129 in enzyme catalysis. The Asp-129 to alanine mutant has been prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. The purified mutant elicited an highly reduced specific activity (about 0.04% of the activity of the wild-type) and a native-like fold, as judged by ¹H NMR spectroscopy. The kinetic analysis revealed that the mutant is able to bind the substrate and a competitive inhibitor, such as inorganic phosphate. Moreover, trapping experiments demonstrated it maintains the ability to form the E-P covalent complex. The Asp-129 to alanine mutant shows extremely reduced enzyme phosphorylation (k₂) and dephosphorylation (k₃) kinetic constant values as compared to the wild-type enzyme. The data reported indicate that aspartic acid-129 is likely to be involved both in the first step and in the rate-limiting step of the catalytic mechanism, i.e. the nucleophilic attack of the phosphorylated intermediate.     

Ribonuclease from Bacillus thuringiensis var. subtoxicus. Gene structure and biosynthesis regulation

The gene for extracellular guanyl-specific ribonuclease of Bacillus thuringiensis var. subtoxicus (RNase Bth), a close homologue of the B. intermedius RNase (binase), was completely sequenced. Analysis of nucleotide sequences in the regions adjoining RNase genes revealed an identical organization of the chromosomal loci of RNase Bth and binase. Growth characteristics of the Bacillus thuringiensis var. subtoxicus strain and its synthesis of RNase were studied. It was shown that the exogenous inorganic phosphate inhibits the biosynthesis of RNase. At the same time, actinomycin D in low doses stimulates the enzyme synthesis. Comparative analysis of the influence of inorganic phosphate and actinomycin D on the biosynthesis of RNAse Bth and binase suggests a possibility of coincidence of regulatory pathways of synthesis of these enzymes.     

Mutational analysis of substrate specificity in a <Emphasis Type="Italic">Citrus paradisi</Emphasis> flavonol 3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

Citrus paradisi 3-O-glucosyltransferase (Cp3GT, Genbank Protein ID: ACS15351) and Citrus sinensis 3-O-glucosyltransferase (Cs3GT, Genbank Protein ID: AAS00612.2) share 95% amino acid sequence identity. Cp3GT was previously established as a flavonol-specific 3-O-glucosyltransferase by direct enzymatic analysis. Cs3GT is annotated as a flavonoid-3-O-glucosyltransferase and predicted to use anthocyanidins as substrates based on gene expression analysis correlated with the accumulation of anthocyanins in C. sinensis cv. Tarocco, a blood orange variety. Mutant enzymes in which amino acids found in Cs3GT were substituted for position equivalent residues in Cp3GT were generated, heterologously expressed in yeast, and characterized for substrate specificity. Structure–function relationships were investigated for wild type and mutant glucosyltransferases by homology modelling using a crystallized Vitis vinifera anthocyanidin/flavonol 3-O-GT (PDB: 2C9Z) as template and subsequent substrate docking. All enzymes showed similar patterns for optimal temperature, pH, and UDP/metal ion inhibition with differences observed in kinetic parameters. Although changes in the activity of the mutant proteins as compared to wild type were observed, cyanidin was never efficiently accepted as a substrate.     

Correlations between kinetic and X-ray analyses of engineered enzymes: crystal structures of mutants Cys----Gly-35 and Tyr----Phe-34 of tyrosyl-tRNA synthetase

The crystal structures of two mutant tyrosyl-tRNA synthetases (TyrTS) are reported to test predictions from kinetic data about structural perturbations and also to aid in the interpretation of apparent strengths of hydrogen bonds measured by protein engineering. The enzyme-tyrosine and enzyme-tyrosyl adenylate complexes of the mutant, TyrTS(Cys----Gly-35), have been determined at 2.5- and 2.7-A resolution, respectively. Residue Cys-35 is in the ribose binding site. Small rearrangements in structure are seen in the enzyme-tyrosine complex that are localized around the cavity created by the mutation. The side chain of Thr-51 moves to occupy the cavity, and Ile-52 adopts two significantly populated conformations, one as in the native enzyme and a second unique to the mutant. On binding tyrosyl adenylate, Ile-52 in the mutant crystal structure preferentially occupies the conformation observed in the native structure. The side chain at Thr-51 becomes disordered. The double-mutant test, which was designed to detect interactions between residues, had previously shown a discrepancy of some 0.4 kcal/mol on mutating Cys-35 and Thr-51 separately and together. A crystal structure of a second mutant, delta TyrTS(Tyr----Phe-34), complexed with tyrosine has been determined at 2.7-A resolution. Tyr-34 in wild-type enzyme makes a hydrogen bond with the phenolic oxygen of the bound tyrosine substrate. The mutant crystal structure was solved to discover whether or not a water molecule binds to the substrate instead of the hydroxyl of Tyr-34 as the interpretation of apparent binding energies from site-directed mutagenesis experiments hinges crucially on whether there is access of water to the mutated region.