Role of acidic amino acid residues in chitooligosaccharide-binding to Streptomyces sp. N174 chitosanase

We examined the oligosaccharide binding to Streptomyces sp. N174 chitosanase by fluorescence spectroscopy. By means of the tryptophan fluorescence quenching, the oligosaccharide binding abilities were evaluated using the three mutant enzymes (D57A, E197A, and D201A). The enzymatic activities of the mutant enzymes were 0.5%, 20.0%, and 38.5% of that of the wild type, respectively. Scatchard plot obtained for the wild type enzyme showed a biphasic profile, suggesting that the oligosaccharide binds to the chitosanase with two different binding sites (the high affinity site and the low affinity site). In contrast, Scatchard plot for E197A exhibited a monophasic profile, in which the slope of the line corresponds to that for the low affinity binding of the wild type enzyme. A monophasic profile was also obtained for D201A, but the slope of the line was similar to that of the high affinity binding. Thus, we conclude that Glu197 and Asp201 are responsible for oligosaccharide binding at the high affinity site and the low affinity site, respectively, which correspond to the (-n) subsites and the (+n) subsites (n=1, 2, and 3). The fluorescence quenching was very weak in D57A, suggesting a strong contribution of this residue to the oligosaccharide binding.     

Mechanism of chitosanase-oligosaccharide interaction: subsite structure of Streptomyces sp. N174 chitosanase and the role of Asp57 carboxylate.

We have investigated the mechanism of the interaction of Streptomyces sp. N174 chitosanase with glucosamine hexasaccharide [(GlcN)(6)] by site-directed mutagenesis, thermal unfolding, and (GlcN)(6) digestion experiments, followed by theoretical calculations. From the energy-minimized model of the chitosanase-(GlcN)(6) complex structure (Marcotte et al., 1996), Asp57, which is present in all known chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (T(m)), usually observed upon the addition of (GlcN)(n) to chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)(6)] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.     

Purification and characterization of a chitosanase from Streptomyces N174

A highly efficient chitosanase producer, the actinomycete N174, identified by chemotaxonomic methods as belonging to the genus Streptomyces was isolated from soil. Chitosanase production by N174 was inducible by chitosan or d-glucosamine. In culture filtrates the chitosanase accounted for 50–60% of total extracellular proteins. The chitosanase was purified by polyacrylic acid precipitation, CM-Sepharose and gel permeation chromatography. The maximum velocity of chitosan degradation was obtained at 65° C when the pH was maintained at 5.5. The enzyme degraded chitosans with a range of acetylation degrees from 1 to 60% but not chitin or CM-cellulose. The enzyme showed an endo-splitting type of activity and the end-product of chitosan degradation contained a mixture of dimers and trimers of d-glucosamine.Correspondence to: R. Brzezinski     

The molecular surface of proteolytic enzymes has an important role in stability of the enzymatic activity in extraordinary environments.

It is scientifically and industrially important to clarify the stabilizing mechanism of proteases in extraordinary environments. We used subtilisins ALP I and Sendai as models to study the mechanism. Subtilisin ALP I is extremely sensitive to highly alkaline conditions, even though the enzyme is produced by alkalophilic Bacillus, whereas subtilisin Sendai from alkalophilic Bacillus is stable under conditions of high alkalinity. We constructed mutant subtilisin ALP I enzymes by mutating the amino acid residues specific for subtilisin ALP I to the residues at the corresponding positions of amino acid sequence alignment of alkaline subtilisin Sendai. We observed that the two mutations in the C-terminal region were most effective for improving stability against surfactants and heat as well as high alkalinity. We predicted that the mutated residues are located on the surface of the enzyme structures and, on thebasis of three-dimensional modelling, that they are involved in stabilizing the conformation of the C-terminal region. As proteolytic enzymes frequently become inactive due to autocatalysis, stability of these enzymes in an extraordinary environment would depend on the conformational stability of the molecular surface concealing scissile peptide bonds. It appeared that the stabilization of the molecular surface structure was effective to improve the stability of the proteolytic enzymes.     

Val65 plays an important role in the substrate synergism, structural stability and activity of arginine kinase

Arginine kinase, a member of phosphagen kinase, is a key enzyme in the cellular energy metabolism of invertebrates. A series mutation of conserved amino acid residue V65 was constructed to investigate its role in AK substrate synergism, structural stability and activity. Our study revealed that mutation in this conserved site could cause pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Spectroscopic experiments indicated that these mutations influenced transition from the molten globule intermediate to the native state in folding process. These results provided herein suggest that amino acid residue V65 played a relatively important role in AK substrate synergism, structural stability and activity.     

Polymer stability plays an important role in the positional regulation of FtsZ

We conducted a series of experiments examining the effect of polymer stability on FtsZ localization dynamics in Bacillus subtilis. A loss-of-function mutation in ezrA, a putative polymer-destabilizing factor, suppresses the defects in FtsZ polymer stability associated with minCD overexpression. In addition, a mutation that is predicted to stabilize the FtsZ polymer leads to the formation of polar FtsZ rings. These data support the hypothesis that carefully balanced polymer stability is important for the assembly and localization of FtsZ during the bacterial cell cycle.     

Alpha7 helix plays an important role in the conformational stability of PTP1B

The C-terminus of Protein Tyrosine Phosphatase 1B (PTP1B) includes an α-helix α7), which forms an allosteric binding site 20 ? away from the active site. This helix is specific to PTP1B and its truncation decreases the catalytic activity significantly. Here, molecular dynamics (MD) simulations in the presence and absence of α7 were performed to investigate the role played by α7. The highly mobile α7 was found to maintain its contacts with loop 11 (L11)α3 helix throughout the simulations. The interactions of Tyr152 on L11, Tyr176, Thr177 on the catalytically important WPD loop and Ser190 on α3 are important for the conformational stability and the concerted motions of the regions surrounding the WPD loop. In the absence of α7, L11 and WPD loop move away from their crystal structure conformations, resulting in the loss of the interactions in this region, and a decrease in the residue displacement correlations in the vicinity of WPD loop. Therefore, we suggest that one of the functionally important roles of α7 may be to limit the L11 and α3 motions, and, facilitate the WPD loop motions. Truncation of α7 in PTP1B is found to affect distant regions as well, such as the substrate recognition site and the phosphate binding-loop (P-loop), changing the conformations of these regions significantly. Our results show that the PTP1B specific α7 is important for the conformation and dynamics of the WPD loop, and also may play a role in ligand binding.     

The role of Arg46 and Arg47 of antithrombin in heparin binding.

Heparin greatly accelerates the reaction between antithrombin and its target proteinases, thrombin and factor Xa, by virtue of a specific pentasaccharide sequence of heparin binding to antithrombin. The binding occurs in two steps, an initial weak interaction inducing a conformational change of antithrombin that increases the affinity for heparin and activates the inhibitor. Arg46 and Arg47 of antithrombin have been implicated in heparin binding by studies of natural and recombinant variants and by the crystal structure of a pentasaccharide-antithrombin complex. We have mutated these two residues to Ala or His to determine their role in the heparin-binding mechanism. The dissociation constants for the binding of both full-length heparin and pentasaccharide to the R46A and R47H variants were increased 3-4-fold and 20-30-fold, respectively, at pH 7.4. Arg46 thus contributes only little to the binding, whereas Arg47 is of appreciable importance. The ionic strength dependence of the dissociation constant for pentasaccharide binding to the R47H variant showed that the decrease in affinity was due to the loss of both one charge interaction and nonionic interactions. Rapid-kinetics studies further revealed that the affinity loss was caused by both a somewhat lower forward rate constant and a greater reverse rate constant of the conformational change step, while the affinity of the initial binding step was unaffected. Arg47 is thus not involved in the initial weak binding of heparin to antithrombin but is important for the heparin-induced conformational change. These results are in agreement with a previously proposed model, in which an initial low-affinity binding of the nonreducing-end trisaccharide of the heparin pentasaccharide induces the antithrombin conformational change. This change positions Arg47 and other residues for optimal interaction with the reducing-end disaccharide, thereby locking the inhibitor in the activated state.     

Identification of critical residues for the activity and thermostability of Streptomyces sp. SK glucose isomerase

The role of residue 219 in the physicochemical properties of d-glucose isomerase from Streptomyces sp. SK strain (SKGI) was investigated by site-directed mutagenesis and structural studies. Mutants G219A, G219N, and G219F were generated and characterized. Comparative studies of their physicochemical properties with those of the wild-type enzyme highlighted that mutant G219A displayed increased specific activity and thermal stability compared to that of the wild-type enzyme, while for G219N and G219F, these properties were considerably decreased. A double mutant, SKGI F53L/G219A, displayed a higher optimal temperature and a higher catalytic efficiency than both the G219A mutant and the wild-type enzyme and showed a half-life time of about 150 min at 85 °C as compared to 50 min for wild-type SKGI. Crystal structures of SKGI wild-type and G219A enzymes were solved to 1.73 and 2.15 Å, respectively, and showed that the polypeptide chain folds into two structural domains. The larger domain consists of a (β/α)₈ unit, and the smaller domain forms a loop of α helices. Detailed analyses of the three-dimensional structures highlighted minor but important changes in the active site region as compared to that of the wild-type enzyme leading to a displacement of both metal ions, and in particular that in site M2. The structural analyses moreover revealed how the substitution of G219 by an alanine plays a crucial role in improving the thermostability of the mutant enzyme.     

Short communication: Production of l-glutamate oxidase by Streptomyces sp. N1 in submerged fermentation

In submerged fermentation of Streptomyces sp. N1 in a shake flask, glucose (3% w/v) and (NH₄)₂SO₄ (0.6% w/v) were found to be suitable for extracellular l-glutamate oxidase (GluOx) (EC.1.4.3.11) production. GluOx production was higher with the addition of further KCl or MgCl₂ to the medium within the range of 0 to 0.12% (w/v). The effect of inoculum type, that is, spore inoculation or mycelium inoculation on GluOx biosynthesis was also investigated, and the maximum GluOx production obtained was 2.7 U/ml after 33h fermentation with mycelium inoculation. The results demonstrated a much higher GluOx production and productivity compared with those reported previously.     

Histidine-15: an important role in the cytotoxic activity of human tumor necrosis factor

The amino acids that are required for the cytotoxic activity of recombinant human tumor necrosis factor-alpha (TNF) were investigated by chemical modification and oligonucleotide-directed site-specific mutagenesis. TNF contains three histidine residues, located at positions 15, 73 and 78. The histidine-specific reagent diethylpyrocarbonate (DEP) was used to chemically modify TNF. The chemical inactivation of the in vitro cytotoxic activity of this lymphokine (using murine L929 target cells) was found to be time- and dose-dependent. Inactivated TNF failed to compete with fully bioactive [125I]TNF for human MCF-7 target cell receptors. Mutant polypeptides of TNF were genetically engineered by oligonucoleotide-directed site-specific mutagenesis. The cytotoxicity of a double histidine mutant, in which histidine-73 and histidine-78 were replaced with glutamine, was not altered and was chemically inactivated by DEP. Substituting glutamine for histidine-15 resulted in 10-15% of the wild-type bioactivity. Replacing histidine-15 with either asparagine, lysine or glycine resulted in a biologically inactive molecule. The data show that the histidine residue at position 15 is an amino acid that is required for the cytotoxic activity of TNF.     

Identification of Streptomyces sp. KH29, which produces an antibiotic substance processing an inhibitory activity against multidrug-resistant Acinetobacter baumannii

The Actinomycete strain KH29 is antagonistic to the multidrug-resistant Acinetobacter baumannii. Based on the diaminopimelic acid (DAP) type, and the morphological and physiological characteristics observed through the use of scanning electron microscopy (SEM), KH29 was confirmed as belonging to the genus Streptomyces. By way of its noted 16S rDNA nucleotide sequences, KH29 was found to have a relationship with Streptomyces cinnamonensis. The production of an antibiotic from this strain was found to be most favorable when cultured with glucose, polypeptone, and yeast extract (PY) medium for 6 days at 27 degrees C. The antibiotic produced was identified, through comparisons with reported spectral data including MS and NMR as a cyclo(L-tryptophanyl-L-tryptophanyl). Cyclo(L-Trp-L-Trp), from the PY cultures of KH29, was seen to be highly effective against 41 of 49 multidrugresistant Acinetobacter baumannii. Furthermore, cyclo(LTrp- L-Trp) had antimicrobial activity against Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Saccharomyces cerevisiae, Aspergillus niger, and Candida albicans, However, it was ineffective against Streptomyces murinus.     

Characterization of structural features important for T7 RNAP elongation complex stability reveals competing complex conformations and a role for the non-template strand in RNA displacement.

We have characterized the roles of the phage T7 RNA polymerase (RNAP) thumb subdomain and the RNA binding activity of the N-terminal domain in elongation complex (EC) stability by evaluating how disrupting these structures affects the dissociation rates of halted ECs. Our results reveal distinct roles for these elements in EC stabilization. On supercoiled or partially single-stranded templates the enzyme with a deletion of the thumb subdomain is exceptionally unstable. However, on linear duplex templates the polymerase which has been proteolytically cleaved within the N-terminal domain is the most unstable. The differences in the effects of these RNAP modifications on the stability of ECs on the different templates appear to be due to differences in EC structure: on the linear duplex templates the RNA is properly displaced from the DNA, but on the supercoiled or partially single-stranded templates an extended RNA:DNA hybrid makes a larger contribution to the conformational state of the EC. The halted EC can therefore exist either in a conformation in which the RNA is displaced from the DNA and forms an interaction with the RNAP, or in a conformation in which a more extended RNA:DNA hybrid is present and the RNA:RNAP interaction is less extensive. The partitioning between these competing conformations appears to be a function of the energetics of template reannealing and the relative strengths of the RNA:RNAP interaction and the RNA:DNA hybrid.