Efficient constitutive expression of thermostable 4-α-glucanotransferase in Bacillus subtilis using dual promoters

4-α-Glucanotransferases possess strong transglycosylation activity which has been used in various carbohydrate chemistry fields. Due to safety issues of the recombinant enzymes we chose Bacillus subtilis as an expression host to produce a thermostable 4-α-glucanotransferase from Thermus scotoductus (TSαGT). The HpaII promoter in the Gram-positive bacterial vector pUB110 was used first to express TSαGT gene in B. subtilis. However, the activity of TSαGT in B. subtilis was only 4% of that in our previous Escherichia coli system. Two expression systems constructed by sequential alignment of another constitutive promoter for either α-amylase from B. subtilis NA64 or maltogenic amylase from Bacillus licheniformis downstream of the HpaII promoter elevated the TSαGT productivity by 11- and 12-fold, respectively, compared to the single HpaII promoter system. In conclusion, the dual promoter systems in this study were much better than the single promoter system to express the TSαGT gene in B. subtilis.     

Inhibition of neuraminidase activity by polyphenol compounds isolated from the roots of Glycyrrhiza uralensis

We isolated 18 polyphenols with neuraminidase inhibitory activity from methanol extracts of the roots of Glycyrrhiza uralensis. These polyphenols consisted of four chalcones (1–4), nine flavonoids (5–13), four coumarins (14–17), and one phenylbenzofuran (18). When we tested the effects of these individual compounds and analogs thereof on neuraminidase activation, we found that isoliquiritigenin (1, IC₅₀ = 9.0 μM) and glycyrol (14, IC₅₀ = 3.1 μM) had strong inhibitory activity. Structure–activity analysis showed that the furan rings of the polyphenols were essential for neuraminidase inhibitory activity, and that this activity was enhanced by the apioside group on the chalcone and flavanone backbone. In addition, the presence of a five-membered ring between C-4 and C-2′ in coumestan was critical for neuraminidase inhibition. All neuraminidase inhibitors screened were found to be reversible noncompetitive inhibitors.     

Beta sheet 2–alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein–protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1•CPR complex and inactive (CYP2B1)₂•CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge–charge interactions between CPR and complex partners.     

Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis

Random mutagenesis was performed on β-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K–T307I were approx. 140, 190 and 200%, respectively, of wild type β-agarase (661 U/mg) at 40°C. All three mutant enzymes were stable up to 50°C and E99K–T307I had the highest thermostability. The melting temperature (T _m) of E99K–T307I, determined by CD spectra, was increased by 5.2°C over that of the wild-type enzyme (54.6°C). Activities of both the wild-type and E99K–T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl₂. The E99K–T307I enzyme was stable at 55°C with 1 mM CaCl₂, reaching 260% of the activity the wild-type enzyme held at 40°C without CaCl₂.     

The tyrosine phosphatase, SHP-1, is involved in bronchial mucin production during oxidative stress

Mucus hypersecretion is a clinically important manifestation of chronic inflammatory airway diseases, such as asthma and Chronic obstructive pulmonary disease (COPD). Mucin production in airway epithelia is increased under conditions of oxidative stress. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 suppression is related to the development of airway inflammation and increased ROS levels. In this study, we investigated the role of SHP-1 in mucin secretion triggered by oxidative stress. Human lung mucoepidermoid H292 carcinoma cells were transfected with specific siRNA to eliminate SHP-1 gene expression. Cultured cells were treated with hydrogen peroxide (H₂O₂), and Mucin 5AC(MUC5AC) gene expression and mucin production were determined. Activation of p38 mitogen activated protein kinase (MAPK) in association with MUC5AC production was evaluated. N-acetylcysteine (NAC) was employed to determine whether antioxidants could block MUC5AC production. To establish the precise role of p38, mucin expression was observed after pre-treatment of SHP-1-depleted H292 cells with the p38 chemical blocker. We investigated the in vivo effects of oxidative stress on airway mucus production in SHP-1-deficient heterozygous (mev/+) mice. MUC5AC expression was enhanced in SHP-1 knockdown H292 cells exposed to H₂O₂, compared to that in control cells. The ratio between phosphorylated and total p38 was significantly increased in SHP-1-deficient cells under oxidative stress. Pre-treatment with NAC suppressed both MUC5AC production and p38 activation. Blockage of p38 MAPK led to suppression of MUC5AC mRNA expression. Notably, mucin production was enhanced in the airway epithelia of mev/+ mice exposed to oxidative stress. Our results clearly indicate that SHP-1 plays an important role in airway mucin production through regulating oxidative stress.     

Crystal structures of two archaeal Pelotas reveal inter-domain structural plasticity

Dom34 from Saccharomyces cerevisiae is one of the key players in no-go mRNA decay, a surveillance pathway by which an abnormal mRNA stalled during translation is degraded by an endonucleolytic cleavage. Its homologs called Pelota are found in other species. We showed previously that S. cerevisiae Dom34 (domain 1) has an endoribonuclease activity, which suggests its direct catalytic role in no-go decay. Pelota from Thermoplasma acidophilum and Dom34 from S. cerevisiae have been structurally characterized, revealing a tripartite architecture with a significant difference in their overall conformations. To gain further insights into structural plasticity of the Pelota proteins, we have determined the crystal structures of two archaeal Pelotas from Archaeoglobus fulgidus and Sulfolobus solfataricus. Despite the structural similarity of their individual domains to those of T. acidophilum Pelota and S. cerevisiae Dom34, their overall conformations are distinct from those of T. acidophilum Pelota and S. cerevisiae Dom34. Different overall conformations are due to conformational flexibility of the two linker regions between domains 1 and 2 and between domains 2 and 3. The observed inter-domain structural plasticity of Pelota proteins suggests that large conformational changes are essential for their functions.