Development of subunit selective proteasome substrates for Schistosoma species.

Schistosomiasis, or bilharzia, is a neglected tropical disease caused by Schistosoma spp. blood flukes that infects over 200 million people worldwide. Just one partially effective drug is available, and new drugs and drug targets would be welcome. The 20S proteasome is a validated drug target for many parasitic infections, including those caused by Plasmodium and Leishmania. We previously showed that anticancer proteasome inhibitors that act through the Schistosoma mansoni 20S proteasome (Sm20S) kill the parasite in vitro. To advance these initial findings, we employed Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to define the substrate cleavage specificities of the three catalytic ß subunits of purified Sm20S. The profiles in turn were used to design and synthesize subunit-specific optimized substrates that performed two to eight fold better than the equivalent substrates used to measure the activity of the constitutive human proteasome (c20S). These specific substrates also eliminated the need to purify Sm20S from parasite extracts - a single step enrichment was sufficient to accurately measure substrate hydrolysis and its inhibition with proteasome inhibitors. Finally, we show that the substrate and inhibition profiles for the 20S proteasome from the three medically important schistosome species are similar, suggesting that data arising from an inhibitor development campaign that focuses on Sm20S can be extrapolated to the other two targets with consequent time and cost savings.

Evaluation of application potential of dye-decolorizing peroxidase from Bacillus amyloliquefaciens in bioremediation of paper and pulp mill effluent.

Pulp and paper mill effluent is rich in recalcitrant and toxic pollutants compounds and causes pollution. To find an efficient biocatalyst for the treatment of effluent, a dye-decolorizing peroxidase from Bacillus amyloliquefaciens MN-13, which is capable of degrading lignin, was used for the bioremediation of paper and pulp mill effluent. The dye-decolorizing peroxidase from Bacillus amyloliquefaciens (BaDyP) exhibited high-redox potential to 2, 2'-azinobis (3-ethylbenzothiazoline- 6-sulfonic acid) ammonium salt (ABTS), veratryl alcohol, Mn2+, reactive blue 19, reactive black 5 and lignin dimer guaiacylglycerol-beta-guaiacyl ether (GGE). When GGE was used as substrate, BaDyP broke ß-O-4 bond of GGE and then oxidize Cα to generate vanillin. The Km values for ABTS and veratryl alcohol were 2.19 mm and 0.07 mm, respectively. The Vmax for ABTS and veratryl alcohol were 1.8 mm/min and 14.12 mm/min, respectively. The BaDyP-mediated treatment of pulp and paper mill effluent led to significant reduction of chemical oxygen demand (COD) and color. When 5% (v/v) of effluent was treated with BaDyP for 12 h at 30°C and pH 2, the removal of COD, color, and lignin was achieved at 82.7, 80.2, and 78.20%, respectively. In detoxification assay, the seeds of Vigna unguiculata grown in treated effluent showed a significant increase in germination rate from 66.7% (untreated effluent) to 90%, and in radicle length from 0.68 cm (untreated effluent) to 1.26 cm, respectively. In the meanwhile, the inhibition of Escherichia coli and Bacillus subtilis by the treated effluent reduced significantly as compared to untreated effluent, indicating high detoxification performance of BaDyP for the treatment of pulp and paper mill effluent. The findings suggest that BaDyP is a potential catalyst for bioremediation of pulp and paper mill effluent, as it is effective in substantial lowering of pollutants load as well as reduces COD, color, and toxicity of effluent.

The ergothioneine transporter (ETT): substrates and locations, an inventory.

In all vertebrates including mammals, the ergothioneine transporter ETT (obsolete name OCTN1; human gene symbol SLC22A4) is a powerful and highly specific transporter for the uptake of ergothioneine (ET). ETT is not expressed ubiquitously and only cells with high ETT cell-surface levels can accumulate ET to high concentration. Without ETT, there is no uptake because the plasma membrane is essentially impermeable to this hydrophilic zwitterion. Here, we review the substrate specificity and localization of ETT, which is prominently expressed in neutrophils, monocytes/macrophages, and developing erythrocytes. Most sites of strong expression are conserved across species, but there are also major differences. In particular, we critically analyze the evidence for the expression of ETT in the brain as well as recent data suggesting that the transporter SLC22A15 may also transport ET. We conclude that, to date, ETT remains the only well-defined biomarker for intracellular ET activity. In humans, the ability to take up, distribute, and retain ET depends principally on this transporter.

Biochemical characterization and substrate profiling of a reversible 2,3-dihydroxybenzoic acid decarboxylase for biocatalytic Kolbe-Schmitt reaction.

Reversible benzoic acid decarboxylases are versatile biocatalysts by taking advantage of both decarboxylation and carboxylation reactions, especially for the biocatalytic Kolbe-Schmitt reaction. In the course of developing a benzoic acid decarboxylase tool-box, a putative benzoic acid decarboxylase gene from Fusarium oxysporum was heterologously over-expressed in Escherichia coli, the recombinant protein was purified and characterized. The purified enzyme exhibited relatively high catalytic efficiencies for the decarboxylation of 2, 3-dihydroxybenzoic acid and carboxylation of catechol (kcat/Km = 2.03 × 102 and 1.88 mM-1 min-1, respectively), and thus characterized as 2, 3-dihydroxybenzoic acid decarboxylase (2, 3-DHBD_Fo). The enzyme also catalyzed the decarboxylation of various substituted salicylic acids with different groups at varied positions except 5-position and the carboxylation of phenol and the substituted phenols. In a preparative reaction, catechol was carboxylated into 2, 3-dihydroxybenoic acid with 95% conversion by adding dodecyldimethylbenzylammonium chloride into the reaction system, and the product was isolated in 72% yield. These results demonstrate that 2, 3-DHBD_Fo is a valuable addition to the benzoic acid decarboxylase tool-box with potential practical applications.

Shaping Substrate Selectivity in a Broad-Spectrum Metallo-ß-Lactamase.

Metallo-ß-lactamases (MBLs) are the major group of carbapenemases produced by bacterial pathogens. The design of MBL inhibitors has been limited by, among other issues, incomplete knowledge about how these enzymes modulate substrate recognition. While most MBLs are broad-spectrum enzymes, B2 MBLs are exclusive carbapenemases. This narrower substrate profile has been attributed to a sequence insertion present in B2 enzymes that limits accessibility to the active site. In this work, we evaluate the role of sequence insertions naturally occurring in the B2 enzyme Sfh-I and in the broad-spectrum B1 enzyme SPM-1. We engineered a chimeric protein in which the sequence insertion of SPM-1 was replaced by the one present in Sfh-I. The chimeric variant is a selective cephalosporinase, revealing that the substrate profile of MBLs can be further tuned depending on the protein context. These results also show that the stable scaffold of MBLs allows a modular engineering much richer than the one observed in nature.