Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering

The lacto-N-biosidase LnbB from Bifidobacterium bifidum JCM 1254 was engineered to improve its negligible transglycosylation efficiency with the purpose of enzymatically synthesizing lacto-N-tetraose (LNT; Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc) in one enzymatic step. LNT is a prebiotic human milk oligosaccharide in itself and constitutes the structural core of a range of more complex human milk oligosaccharides as well. Thirteen different LnbB variants were expressed and screened for transglycosylation activity by monitoring transglycosylation product formation using lacto-N-biose 1,2-oxazoline as donor substrate and lactose as acceptor substrate. LNT was the major reaction product, yet careful reaction analysis revealed the formation of three additional LNT isomers, which we identified to have a β1,2-linkage, a β1,6-linkage, and a 1,1-linkage, respectively, between lacto-N-biose (Gal-β1,3-GlcNAc) and lactose. Considering both maximal transglycosylation yield and regioselectivity as well as minimal product hydrolysis, the best variant was LnbB W394H, closely followed by W465H and Y419N. A high transglycosylation yield was also obtained with W394F, yet the substitution of W394 and W465 of the subsite −1 hydrophobic platform in the enzyme with His dramatically impaired the undesirable product hydrolysis as compared to substitution with Phe; the effect was most pronounced for W465. Using p-nitrophenyl-β-lacto-N-bioside as donor substrate manifested W394 as an important target position. The optimization of the substrate concentrations confirmed that high initial substrate concentration and high acceptor-to-donor ratio both favor transglycosylation.

Different Oxidation Pathways of 2-Selenouracil and 2-Thiouracil, Natural Components of Transfer RNA

Sulfur- and selenium-modified uridines present in the wobble position of transfer RNAs (tRNAs) play an important role in the precise reading of genetic information and tuning of protein biosynthesis in all three domains of life. Both sulfur and selenium chalcogens functionally operate as key elements of biological molecules involved in the protection of cells against oxidative damage. In this work, 2-thiouracil (S2Ura) and 2-selenouracil (Se2Ura) were treated with hydrogen peroxide at 1:0.5, 1:1, and 1:10 molar ratios and at selected pH values ranging from 5 to 8. It was found that Se2Ura was more prone to oxidation than its sulfur analog, and if reacted with H2O2 at a 1:1 or lower molar ratio, it predominantly produced diselenide Ura-Se-Se-Ura, which spontaneously transformed to a previously unknown Se-containing two-ring compound. Its deselenation furnished the major reaction product, a structure not related to any known biological species. Under the same conditions, only a small amount of S2Ura was oxidized to form Ura-SO2H and uracil (Ura). In contrast, 10-fold excess hydrogen peroxide converted Se2Ura and S2Ura into corresponding Ura-SeOnH and Ura-SOnH intermediates, which decomposed with the release of selenium and sulfur oxide(s) to yield Ura as either a predominant or exclusive product, respectively. Our results confirmed significantly different oxidation pathways of 2-selenouracil and 2-thiouracil.

Analysis of a cellulose synthase catalytic subunit from the oomycete pathogen of crops Phytophthora capsici

Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes.

One-pot synthesis of substituted pyrrolo[3,4-b]pyridine-4,5-diones based on the reaction of N-(1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-2-oxo-2-arylethyl)acetamide with amines

The condensation of primary amines with N-(1-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-2-oxo-2-arylethyl)acetamides was explored. Thus, a previously unknown recyclization of the starting material was observed in acidic ethanol in the presence of an amine, which provided the corresponding dihydropyrrolone derivative as the major reaction product. Based on this transformation, a practical and convenient one-pot synthetic method for substituted pyrrolo[3,4-b]pyridin-5-ones could be devised.

Daurichromenic Acid-producing Oxidocyclase in the Young Leaves of Rhododendron dauricum

Rhododendron dauricum L., a flowering tree popular in Hokkaido, produces daurichromenic acid (DCA), a terpenophenol with a potent anti-HIV activity. The DCA-producing enzyme, named DCA synthase, could be detected in the soluble protein fraction prepared from the young leaves of R. dauricum. DCA synthase catalyzed oxidocyclization of the farnesyl group of grifolic acid to form (+)-DCA as the major reaction product. The DCA synthase reaction proceeds without the need for any cofactors and coenzymes except for molecular oxygen. Interestingly, these catalytic properties of DCA synthase are quite similar to those reported for cannabinoid synthases in the marijuana plant Cannabis sativa L.

Reaction Products of Lime Zeolite Stabilized Kaolin Humic Acid

Lime, a traditional calcium based stabilizer, had been widely used in chemical stabilization to improve the strength of soil. Past researches had shown that the major reaction product of lime and soil such as Calcium Silicate Hydrate (CSH) was formed abundantly under the observation of microscopic studies. However, sometimes it will be quite difficult to confirm the existence of CSH phase if solely based on its needle like structures, especially when other rod like structures will also exist. Practically, the recognition of the CSH phase by using XRD spectrum through matching with published data had speed up the process of identification. If the method is viable, then theoretically, the molecular weight ratio of silica and calcium, S/C of CSH gel is specific and can be determined based on its possible chemical compound. Hence, this study was carried out in an attempt to examine the possibility use of its S/C ratio as a quick method to confirm the existence of CSH gel. Two types of artificial organic soils were formed by admixing kaolin (inorganic matter) and humic acid (organic matter) with the ratio of 7:3 and 5:5. Four types of admixtures with different percentages ratio of lime and zeolite (a kind of pozzolan) were used to stabilize the soils. The specimens were cured at elevated temperature of 50°c in order to accelerate the development of reaction products. Field Emission Scanning Electron Microscope with attached Energy Dispersive Analyzer (FESEM-EDX) was utilized to observe and determine the existence of reaction products and its bulk chemical composition. The S/C ratio of needle like structures were determined and it is found that the S/C ratio fluctuates and varies significantly from one specimen to another. It is believed that due to the limitations of the experimental setup, the EDX analysis can only serve as semi-quantitative and act as a reference guide on the existence of element. Despite of its limitations, the EDX analysis is useful in distinguish the CSH from other structure which is physically un-identical.

A Study of the Reaction Between Quinone and 2R4F Cigarette Smoke Condensate

A study using atomic emission detection (AED) as an approach to explore the fate of quinone added into 2R4F cigarette smoke condensate (CSC) have been performed. Both natural isotope quinone and 13C labeled quinone were used in the study. When coupled with a gas chromatographic separation (GC/AED), the AED provided informative new data on 13C isotope enriched products generated following reactions between 2R4F CSC and the quinone. Two 13C containing species were detected by GC/AED. Matching chromatographic separation using gas chromatography/mass selective detection (GC/MSD) allowed for a structural assignment of a relatively minor CSC 13C 6quinone reaction product as nitrohydroquinone (13C6NO2HQ). The chemical mechanism accounting for the formation of 13C6NO2HQ in the CSC was envisioned to be a reaction product between HONO and 13C 6Quinone (13C6Q) to form 13C6NO2Q, followed by reduction of 13C6NO2Q to 13C6NO2HQ. The amount of 13C6NO2HQ accounted for ~6% of the added 13C6Q. Identical trends in reaction chemistries were found for experiments with 12C6Q. The major reaction product detected upon addition of 13C6Q to the 2R4F CSC sample was 13C6HQ. 13C6HQ accounted for, on average, ~47% of the initial 13C6Q concentration. Identical trends in reaction chemistries were found for experiments with 12C6Q. No additional 13C containing species were detected. A 13C AED compound independent calibration (CIC) approach under the operating conditions was not possible. This work further expands the knowledge regarding possible reactions of quinone and hydroquinone in CSC.

Relationship between Antimalarial Activity and Heme Alkylation for Spiro- and Dispiro-1,2,4-Trioxolane Antimalarials

ABSTRACT The reaction of spiro- and dispiro-1,2,4-trioxolane antimalarials with heme has been investigated to provide further insight into the mechanism of action for this important class of antimalarials. A series of trioxolanes with various antimalarial potencies was found to be unreactive in the presence of Fe(III) hemin, but all were rapidly degraded by reduced Fe(II) heme. The major reaction product from the heme-mediated degradation of biologically active trioxolanes was an alkylated heme adduct resulting from addition of a radical intermediate. Under standardized reaction conditions, a correlation (R 2 = 0.88) was found between the extent of heme alkylation and in vitro antimalarial activity, suggesting that heme alkylation may be related to the mechanism of action for these trioxolanes. Significantly less heme alkylation was observed for the clinically utilized artemisinin derivatives compared to the equipotent trioxolanes included in this study.