A Single Residue Influences the Reaction Mechanism of Ammonia Lyases and Mutases

All ways lead to Rome? Computer modeling and kinetic measurements identified a distinct residue in Phe/Tyr ammonia lyases (PAL/TAL) which controls whether the Friedel–Crafts or an E₁cB reaction mechanism takes place. Hence, Glu484 in pcPAL favors the Friedel–Crafts reaction (see picture, MIO=4‐methylidene imidazol‐5‐one) whereas an Asn in TAL gives an elimination reaction. These mechanistic investigations also reveal activity of a PAL mutant and a TAL towards an amino alcohol.

     

The substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata

In previous work, we studied and reported that an enzyme from Curvularia lunata 3.4381 had the novel specificity to hydrolyze the terminal rhamnosyl at C-3 position of steroidal saponin and obtained four transformed products; the enzyme was purified and ascertained as glucoamylase (EC 3.2.1.3 GA). In this work, the enzyme exhibiting steroidal saponin-rhamnosidase activity was systematically studied on 21 steroidal saponins and 6 ginsenosides. The results showed that the α-1,2-linked end-rhamnosyl residues at C-3 position of steroidal saponins could be hydrolyzed to corresponding secondary steroidal saponins, among which 18 compounds were isolated and identified, including 3 new secondary compounds. For the furostanosides having glucosyl residues at the C-26 position, hydrolysis occurred first at end-rhamnosyl at C-3 position to produce secondary furostanosides. The reaction of hydrolyzing glucosyl at C-26 position depended considerably on longer reaction times yielding the corresponding secondary spirostanosides (without rhamnosyl and glucosyl residues). The enzyme had the strict specificity for the terminal α-1,2-linked rhamnosyl residues of linear chain, or the terminal α-1,2-linked rhamnosyl residues with branched chain of 1,4-linked glycosyl residues of sugar chain at C-3 position of steroidal saponins, it was not specific for different aglycones, different glycons, and the number of glycon of sugar chain of steroidal saponin. The end-rhamnosyl of ginsenosides and p-nitrophenyl-α-l-rhamnopyranoside (pNPR) could not be hydrolyzed by the enzyme from C. lunata.     

What a Difference an OH Makes: Conformational Dynamics as the Basis for the Ligand Specificity of the Neomycin‐Sensing Riboswitch

To ensure appropriate metabolic regulation, riboswitches must discriminate efficiently between their target ligands and chemically similar molecules that are also present in the cell. A remarkable example of efficient ligand discrimination is a synthetic neomycin‐sensing riboswitch. Paromomycin, which differs from neomycin only by the substitution of a single amino group with a hydroxy group, also binds but does not flip the riboswitch. Interestingly, the solution structures of the two riboswitch–ligand complexes are virtually identical. In this work, we demonstrate that the local loss of key intermolecular interactions at the substitution site is translated through a defined network of intramolecular interactions into global changes in RNA conformational dynamics. The remarkable specificity of this riboswitch is thus based on structural dynamics rather than static structural differences. In this respect, the neomycin riboswitch is a model for many of its natural counterparts.     

Characterization of a novel anti-human TNF-alpha murine monoclonal antibody with high binding affinity and neutralizing activity

In order to develop an anti-human TNF-alpha mAb, mice were immunized with recombinant human TNF-alpha. A murine mAb, TSK114, which showed the highest binding activity for human TNF-alpha was selected and characterized. TSK114 specifically bound to human TNF-alpha without cross-reactivity with the homologous murine TNF-alpha and human TNF-beta. TSK114 was found to be of IgG1 isotype with kappa light chain. The nucleotide sequences of the variable regions of TSK114 heavy and light chains were determined and analyzed for the usage of gene families for the variable (V), diversity (D), and joining (J) segments. Kinetic analysis of TSK114 binding to human TNF-alpha by surface plasmon resonance technique revealed a binding affinity (K(D)) of approximately 5.3 pM, which is about 1,000- and 100-fold higher than those of clinically relevant infliximab (Remicade) and adalimumab (Humira) mAbs, respectively. TSK114 neutralized human TNF-alpha-mediated cytotoxicity in proportion to the concentration, exhibiting about 4-fold greater efficiency than those of infliximab and adalimumab in WEHI 164 cells used as an in vitro model system. These results suggest that TSK114 has the potential to be developed into a therapeutic TNF-alpha-neutralizing antibody with picomolar affinity.     

Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach

The proteins within the CAZy glycoside hydrolase family GH13 catalyze the hydrolysis of polysaccharides such as glycogen and starch. Many of these enzymes also perform transglycosylation in various degrees, ranging from secondary to predominant reactions. Identifying structural determinants associated with GH13 family reaction specificity is key to modifying and designing enzymes with increased specificity towards individual reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational approach for decoding the determinant structural composition defining the reaction specificity. This method is based on the conservation of coevolving residues in spatial contacts associated with reaction specificity. To evaluate the algorithm, mutants of α-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima were constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio while the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations of the variants indicated changes in flexibility that can account for the modified T/H ratio. An essential contribution of the presented computational approach is its capacity to identify residues outside of the active center that affect the reaction specificity.     

Stress-Relieving Effects of Sesame Oil Aroma and Identification of the Active Components

(1) Sesame oil aroma has stress-relieving properties, but there is little information on its effective use and active ingredients. (2) Methods: ICR male mice were housed under water-immersion stress for 24 h. Then, the scent of sesame oil or a typical ingredient was inhaled to the stress groups for 30, 60, or 90 min. We investigated the effects of sesame oil aroma on mice behavior and the expression of the dual specificity phosphatase 1 (DUSP1) gene, a candidate stress marker gene in the brain. (3) Results: In an elevated plus-maze test, the rate of entering into the open arm of a maze and the staying time were increased to a maximum after 60 min of inhalation, but these effects decreased 90 min after inhalation. As for the single component, anxiolytic effects were observed in the 2,5-dimethylpyrazine and 2-methoxy phenol group, but the effect was weakened in the furfuryl mercaptan group. The expression levels of DUSP1 in the hippocampus and striatum were significantly decreased in 2,5-dimethylpyrazine and 2-methoxy phenol groups. (4) Conclusions: We clarified the active ingredients and optimal concentrations of sesame oil for its sedative effect. In particular, 2,5-dimethylpyrazine and 2-methoxy phenol significantly suppressed the stress-induced changes in the expression of DUSP1, which are strong anti-stress agents. Our results suggest that these molecules may be powerful anti-stress agents.     

Characterization of G-quadruplex/hemin peroxidase: substrate specificity and inactivation kinetics

Recently, G-quadruplex/hemin (G4/hemin) complexes have been found to exhibit peroxidase activity, and this feature has been extensively exploited for colorimetric detection of various targets. To further understand and characterize this important DNAzyme, its substrate specificity, inactivation mechanism, and kinetics have been examined by comparison with horseradish peroxidase (HRP). G4/hemin DNAzyme exhibits broader substrate specificity and much higher inactivation rate than HRP because of the exposure of the catalytic hemin center. The inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H(2)O(2) rather than the destruction of G4. Both the inactivation rate and catalytic oxidation rate of G4/hemin DNAzyme depend on the concentration of H(2)O(2), which suggests that active intermediates formed by G4/hemin and H(2)O(2) are the branch point of catalysis and inactivation. Reducing substrates greatly inhibit the inactivation of G4/hemin DNAzyme by rapidly reacting with the active intermediates. A possible catalytic and inactivation process of G4/hemin has been proposed. These results imply a potential cause for the hemin-mediated cellular injury and provide insightful information for the future application of G4/hemin DNAzyme.     

Expression and characterization of a recombinant Fusarium spp. galactose oxidase

The Fusarium spp. (Dactylium dendroides) galactose oxidase was expressed in Aspergillus oryzae and Fusarium venenatum hosts. Under the control of an A. niger α-amylase or a Fusarium trypsin promoter, high level galactose oxidase expression was achieved. The recombinant oxidase expressed in the A. oryzae host was purified and characterized. The purified enzyme had a molecular weight of 66 k Da on sodium dodecyl sulfate-polymerase gel electrophoresis (SDS-PAGE) and 0.4 mol copper atom per mole protein. The stoichiometry increased to 1.2 after a Cu saturation. Based on a peroxidase-coupled assay, the enzyme preparation showed an activity of 440 turnover per second toward d-galactose (0.1 M) at pH7 and 20°C. The enzyme had an optimal temperature of 60°C at pH 6.0 and an activation free Gibbs energy of 33 kJ/mol. A series of d-galactose derivatives was tested as the reducing substrate for the oxidase. The difference in activity was interpreted by the stereospecificity of the oxidase toward the substituents in the pyranose substrate, particularly on the C5 and the cyclic hemiacetal O sites. The recombinan toxidase could act on some galactose-containing polysaccharides, such as guar gum, but was not able to oxidize several common redox compounds that lacked a primary alcohol functional group.