Insights into Glucose-6-phosphate Allosteric Activation of ß-Glucosidase A.

Second-generation ethanol production involves the use of agricultural and forestry waste as feedstock, being an alternative to the first-generation technology as it relies on low-cost abundant residues and does not affect food agriculture. However, the success of second-generation biorefineries relies on energetically efficient processes and effective enzyme cocktails to convert cellulose into fermentable sugars. ß-glucosidases catalyze the last step on the enzymatic hydrolysis of cellulose; however, they are often inhibited by glucose. Previous studies demonstrated that glucose-6-phosphate (G6P) is a positive allosteric modulator of Bacillus polymyxa ß-glucosidase A, improving enzymatic efficiency, providing thermoresistance, and imparting glucose tolerance. However, the precise molecular details of G6P-ß-glucosidase A interactions have not yet been described so far. We investigated the molecular details of G6P binding into B. polymyxa ß-glucosidase A through in silico docking using the site identification by ligand competitive saturation technology followed by site-directed mutagenesis studies, from which an allosteric binding site for G6P was identified. In addition, a mechanistic shift toward the transglycosylation reaction as opposed to hydrolysis was observed in the presence of G6P, suggesting a new role of G6P allosteric modulation of the catalytic activity of ß-glucosidase A.

Effect of xylitol on salivary ß-glucosidase in humans.

Dental biofilm - in which a diverse set of microorganisms are embedded in a complex polysaccharide matrix that adheres to oral components - is one of the most complex microbial communities in the human body. As biofilm formation is related to oral infections, such as caries and periodontal diseases, strategies for biofilm control are crucial for maintaining oral health. Xylitol, a synthetic sugar used as a sucrose substitute, has been shown to reduce biofilm formation. However, its precise mechanism of action on biofilm reduction has so far not been elucidated. Previous studies demonstrate that bacterial ß-glucosidase action is crucial for biofilm formation. Here, we investigated the correlation between salivary ß-glucosidase activity and dental plaque occurrence. We found a positive correlation between enzymatic activity and the presence of dental biofilm. We observed that xylitol inhibits ß-glucosidase in human saliva. Kinetic studies also confirmed that xylitol acts as a mixed type inhibitor of salivary ß-glucosidase. Based on our data, we suggest that xylitol impairs oral biofilm formation by the inhibition of bacterial ß-glucosidase, which is essential for biofilm formation in the oral cavity.

Beta glucosidase from Bacillus polymyxa is activated by glucose-6-phosphate.

Optimization of cellulose enzymatic hydrolysis is crucial for cost effective bioethanol production from lignocellulosic biomass. Enzymes involved in cellulose hydrolysis are often inhibited by their end-products, cellobiose and glucose. Efforts have been made to produce more efficient enzyme variants that are highly tolerant to product accumulation; however, further improvements are still necessary. Based on an alternative approach we initially investigated whether recently formed glucose could be phosphorylated into glucose-6-phosphate to circumvent glucose accumulation and avoid inhibition of beta-glucosidase from Bacillus polymyxa (BGLA). The kinetic properties and structural analysis of BGLA in the presence of glucose-6-phosphate (G6P) were investigated. Kinetic studies demonstrated that enzyme was not inhibited by G6P. In contrast, the presence of G6P activated the enzyme, prevented beta glucosidase feedback inhibition by glucose accumulation and improved protein stability. G6P binding was investigated by fluorescence quenching experiments and the respective association constant indicated high affinity binding of G6P to BGLA. Data reported here are of great impact for future design strategies for second-generation bioethanol production.

Contribution of light chain residues to high affinity binding in an HIV-1 antibody explored by combinatorial scanning mutagenesis.

Detailed analysis of factors governing high affinity antibody-antigen interactions yields important insight into molecular recognition and facilitates the design of functional antibody libraries. Here we describe comprehensive mutagenesis of the light chain complementarity determining regions (CDRs) of HIV-1 antibody D5 (which binds its target, "5-Helix", with a reported K(D) of 50 pM). Combinatorial scanning mutagenesis libraries were prepared in which CDR residues on the D5 light chain were varied among WT side chain identity or alanine. Selection of these libraries against 5-Helix and then sequence analysis of the resulting population were used to quantify energetic consequences of mutation from wild-type to alanine (DeltaDeltaG(Ala-WT)) at each position. This analysis revealed several hotspot residues (DeltaDeltaG(Ala-WT) >or= 1 kcal/mol) that formed combining site features critical to the affinity of the interaction. Tolerance of D5 light chain residues to alternative mutations was explored with a second library. We found that light chain residues located at the center and at the periphery of the D5 combining site contribute to shape complementarity and electrostatic characteristics. Thus, the affinity of D5 for 5-Helix arises from extended interactions involving both the heavy and light chains of D5. These results provide significant insight for future antibody engineering efforts.

A small-molecule inhibitor of BCL6 kills DLBCL cells in vitro and in vivo.

The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma (DLBCL). We combined computer-aided drug design with functional assays to identify low-molecular-weight compounds that bind to the corepressor binding groove of the BCL6 BTB domain. One such compound disrupted BCL6/corepressor complexes in vitro and in vivo, and was observed by X-ray crystallography and NMR to bind the critical site within the BTB groove. This compound could induce expression of BCL6 target genes and kill BCL6-positive DLBCL cell lines. In xenotransplantation experiments, the compound was nontoxic and potently suppressed DLBCL tumors in vivo. The compound also killed primary DLBCLs from human patients.

Sequential transcription factor targeting for diffuse large B-cell lymphomas.

Transcription factors play a central role in malignant transformation by activating or repressing waves of downstream target genes. Therapeutic targeting of transcription factors can reprogram cancer cells to lose their advantages in growth and survival. The BCL6 transcriptional repressor plays a central role in the pathogenesis of diffuse large B-cell lymphomas (DLBCL) and controls downstream checkpoints, including the p53 tumor suppressor gene. We report that a specific inhibitor of BCL6 called BPI can trigger a p53 response in DLBCL cells. This was partially due to induction of p53 activity and partially due to relief of direct repression by BCL6 of p53 target genes. BPI could thus induce a p53-like response even in the presence of mutant p53. Moreover, sequential BCL6 peptide inhibitors followed by p53 peptide or small-molecule activators provided a more powerful antilymphoma effect than either treatment alone by maximally restoring p53 target gene expression. Therefore, tandem targeting of the overlapping BCL6 and p53 transcriptional programs can correct aberrant survival pathways in DLBCL and might provide an effective therapeutic approach to lymphoma therapy.

Specific peptide interference reveals BCL6 transcriptional and oncogenic mechanisms in B-cell lymphoma cells.

The BTB/POZ transcriptional repressor and candidate oncogene BCL6 is frequently misregulated in B-cell lymphomas. The interface through which the BCL6 BTB domain mediates recruitment of the SMRT, NCoR and BCoR corepressors was recently identified. To determine the contribution of this interface to BCL6 transcriptional and biological properties, we generated a peptide that specifically binds BCL6 and blocks corepressor recruitment in vivo. This inhibitor disrupts BCL6-mediated repression and establishment of silenced chromatin, reactivates natural BCL6 target genes, and abrogates BCL6 biological function in B cells. In BCL6-positive lymphoma cells, peptide blockade caused apoptosis and cell cycle arrest. BTB domain peptide inhibitors may constitute a novel therapeutic agent for B-cell lymphomas.