Conjugation of phosphonoacetic acid to nucleobase promotes a mechanism-based inhibition

Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.     

Cloning and Expression of the Exo-β-D-1,3-glucanase Gene (exgS) from Aspergillus saitoi

A gene of exo-1,3-β-D-glucanase (exgS) was cloned from a koji mold, Aspergillus saitoi, genomic DNA using PCR. The exgS has an ORF comprising 2832 bp, which contains one intron of 45 bp, and encodes 945 amino acids. The deduced amino acid sequences showed that the ExgS has a non-homologous linker region consisting of 180 amino acids, which encompassed highly conserved regions observed in Exg homologues from filamentous fungi. A recombinant protein (ExgS) has been recovered from the cultural filtrate of an Aspergillus oryzae strain that carried an expression vector containing full length of the exgS. The N-terminal amino acid sequences of the recombinant exo-1,3-β-D-glucanase (ExgS) were identical to that of native ExgS from A. saitoi.     

Enzymatic fragmentation of carbohydrate moieties of radish arabinogalactan-protein and elucidation of the structures

We investigated the structures of L-arabino-galactooligosaccharides released from the sugar moieties of a radish arabinogalactan-protein (AGP) by the action of exo-β-(1→3)-galactanase. We detected a series of neutral β-(1→6)-linked galactooligosaccharides forming branches of one to up to at least 19 consecutive Gal groups, together with corresponding acidic derivatives terminating in 4-O-methyl-glucuronic acid (4-Me-GlcA) at the non-reducing end. Some oligosaccharide chains of degree of polymerization (dp) higher than 3 for neutral, and 4 for acidic oligomers were modified with L-Araf residues. The acidic tetrasaccharide 4-Me-β-GlcA-(1→6)[α-L-Araf-(1→3)]-β-Gal-(1→6)-Gal was detected as an abundant L-Araf-containing oligosaccharide among these neutral and acidic oligomers. A pentasaccharide containing an additional L-Araf group attached to the L-Ara in the tetrasaccharide through an α-(1→5)-linkage was also found. We observed L-arabino-galactooligosaccharides substituted with single or disaccharide L-Araf units at different Gal residues along these neutral and acidic β-(1→6)-galactooligosaccharide chains, indicating that these side chains are highly variable in length and substituted variously with L-Araf residues.     

Production of Higher-quality Plastein from a Crude Single-cell Protein

From defatted n-paraffin-assimilating yeast cells, a crude protein was obtained by alkaliextraction followed by acid-precipitation. Then the protein was treated with ether until extractable substances were removed exhaustively at this stage. However, at the next stage where the ether-treated protein had been partially hydrolyzed with pepsin, when the hydrolysate was retreated with ether, it was found that ether-extractable substances totalling 270 mg/100 g were obtainable additionally. Chromatographic investigations demonstrated that the substances included significant amounts of aliphatic and aromatic hydrocarbons, some indoles, and a ubiquinone (n = 8).

From the protein hydrolysate (substrate) after the above ether-treatment, a plastein was synthesized with Bioprase under the specific conditions. The plastein was obtained as a precipitate when the whole reaction mixture was treated with aqueous ethanol or acetone. The quantity and quality (nitrogen content) of the plastein depended on the ethanol or acetone concentration. Roughly speaking, the higher the concentration, the more the plastein quantity. The converse relation held for the quality; a plastein precipitated by treatment solely with water showed a higher quality than any other case.     

Facile One-Pot Three Component Synthesis,Characterization, and Molecular Docking Simulations of Novel α-Aminophosphonate Derivatives Based Pyrazole Moiety as Potential Antimicrobial Agent

An efficient method has been developed for the synthesis of novel α-aminophosphonates (AAP) ( 3 a – m ) through a one-pot three-component reaction of 1,3-disubstituted-1H-pyrazol-5-amine, aromatic aldehydes, and phosphite using lithium perchlorate as catalyst. All newly synthesized compounds were characterized via different spectroscopic techniques. The synthesized compounds′ mode of action was investigated using molecular docking against the outer membrane protein A (OMPA) and exo-1,3-β-glucanase, with interpreting their pharmacokinetics aspects. The results of the antimicrobial effectiveness of these compounds revealed a broad spectrum of their biocidal activity and this in-vitro study was in line with the in- silico results. Additionally, it has been demonstrated that these compounds exhibited a minimum inhibitory concentration (MIC) with significant activity at low concentrations (7.5–30.0 mg/mL). Further, the radical scavenging (DPPH^*) activity of the synthesized compounds fluctuated, with compounds 3 h , 3 a , and 3 f showing the highest antioxidant activity. Overall, the formulated compounds can be employed as antimicrobial and antioxidant agents in medical applications.     

Effect of exo-16,17-dihydro-gibberellin A5 on gibberellin A20 metabolism in seedlings of dwarf rice (Oryza sativa L. cv. Tan-ginbozu)

Several of the 16,17-dihydro gibberellins (GAs) inhibit elongation in a variety of species. In a study of their mechanism of action we have investigated the effect of exo-16,17-dihydro-Ga₅ (diHGA₅) on the metabolism of GA₂₀ in dwarf rice (Oryza sativa cv. Tan-ginbozu). A mixture of [³H]- and [³H]-GA₂₀ (100 ng per plant) was applied in microdrops to 4 d old seedlings which were harvested 72 h later. Concurrent treatment with diHGA₅ at 100 ng or 333 ng per plant reduced GA₂₀-induced elongation of the second leaf sheath by 41–66%. There was a concomitant reduction in the amount of [²H₂]GA₁ present at harvest, measured by gas chromatography-mass spectrometry-selected ion monitoring. The [²H₂]GA₂₉ content was also reduced. There was no clear effect of diHGA₅ on the total radioactivity recovered, or on conversion of the [³H]GA₂₀ to putative [³H]GA conjugates, or on the amount of [²H₂]GA₂₀ found. No free [²H₂]GA₈ was detected. In other experiments there was little effect of diHGA₅ on elongation induced by treatment with GA₁. We conclude that diHGA₅ inhibited GA₂₀-induced elongation in dwarf rice shoots at least partly by reducing the 3-hydroxylation of GA₂₀ to GA₁.Abbreviations diHGA₅ = exo- 16, 17-dihydro-gibberellin A₅ - GA = gibberellin - GC-MS-SIM = gas chromatography-mass spectrometry-selected ion monitoring     

An Asymmetric Synthesis of 2-Substituted Piperidines through Ozonolysis of Cyclopentenes and Reductive Aminocyclization

By the action of ozone, sodium cyanoborohydride and the optically active benzylic amines 2, the 1-substituted cyclopentenes 1, 5 and 9 were converted to a diastereoisomeric mixture of 1,2-disubstituted piperidines (3, 6 and 10), respectively. Hydrogenation of these compounds and the following work-up yielded optically active 2-alkylpiperidines (4, up to 68% e.e.), pipecolic acid (7, 84%e.e.) and 2-(hydroxymethyl)piperidine (11, up to 85%e.e.). Chromatographic separation of the major isomers of 3b and 6 enabled optically pure coniine (4b) and pipecolic acid (7) to be prepared, respectively.     

Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) β-glucosidase from Aspergillus niger ASKU28

Background

The commercially important glycoside hydrolase family 3 (GH3) β-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined.

Methods

In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 β-glucosidase from A. niger ASKU28.

Results

In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced k_cat and k_cat/K_m values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl β-d-glucoside and 2,4-dinitrophenyl β-d-glucoside, the aglycone leaving groups of which are less dependent on acid catalysis, although changes in the rate-determining catalytic step were revealed for both. pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl β-glucosides and yielded β-glucosyl azide as a product.

Conclusions and general significance

These results strongly support the assignment of E490 as the acid/base catalyst in a β-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members.     

Photosystem II Inhibition by s-Triazines Having Hydrophilic Amino Groups

Photosystem II inhibition by s-triazines having hydrophilic amino groups was examined in isolated spinach chloroplasts. Among them, the hydroxyalkyl- and alkoxyalkylamino derivatives were potent inhibitors. The hydroxyalkylamino-s-triazines seemed to interact with the binding site in a manner different from that of other triazines, since they needed a time to build up to constant inhibition and showed a different thermoluminescence glow peak.