Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase

AbstractKinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed 4C1 chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp196, Glu237 and Asp295) and from Arg137. Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7Å) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe52, a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe52 by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp196) are suggested. High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed

Highly Aromatic Flavan-3-ol Derivatives from Palaeotropical <i>Artocarpus lacucha</i> Buch.-Ham Possess Radical Scavenging and Antiproliferative Properties

Phytochemical investigation of leaves and stembark of Artocarpus lacucha collected in Thailand resulted in three yet undescribed isomeric flavan-3-ol derivatives (1–3), the four known compounds gambircatechol (4), (+)-catechin (5), (+)-afzelechin (6) and the stilbene oxyresveratrol (7). Compounds 1 to 3 feature 6/6/5/6/5/6 core structures. All structures were deduced by NMR and MS, while density functional theory (DFT) calculations on B3LYP theory level were performed of compounds 1 to 3 to support the stereochemistry in positions 2 and 3 in the C-ring. Possible biosynthetic pathways leading to 4 are discussed. The DPPH assay revealed high radical scavenging activities for 1 (EC50 = 9.4 ± 1.0 µmol mL−1), 2 (12.2 ± 1.1), 3 (10.0 ± 1.5) and 4 (19.0 ± 2.6), remarkably lower than ascorbic acid (EC50 = 34.9) and α-tocopherol (EC50 = 48.6). A cytotoxicity assay revealed moderate but consistent antiproliferative properties of 1 in CH1/PA-1 (ovarian teratocarcinoma) and SW480 (colon carcinoma) cells, with IC50 values of 25 ± 6 and 34 ± 4 µM, respectively, whereas effects in A549 (non-small cell lung cancer) cells were rather negligible. The performed DCFH-DA assay of 1 in the former cell lines confirmed potent antioxidative effects even in the cellular environment

Phytochemistry Meets Geochemistry&mdash;Blumenol C Sulfate: A New Megastigmane Sulfate from Palicourea&nbsp;luxurians (Rubiaceae: Palicoureeae)

There is a previously neglected influence of geochemical conditions on plant phytochemistry. In particular, high concentrations of dissolved salts can affect their biosynthesis of natural products. Detoxification is most likely an important aspect for the plant, but additional natural products can also give it an expanded range of bioactivities. During the phytochemical analysis a Palicourea luxurians plant collected in a sulfate-rich environment (near the R&iacute;o Sucio, Costa Rica) showed an interesting natural product in this regard. The structure of this compound was determined using spectroscopic and computational methods (NMR, MS, UV, IR, CD, optical rotation, quantum chemical calculations) and resulted in a megastigmane sulfate ester possessing a &beta;-ionone core structure, namely blumenol C sulfate (1, C13H22O5S). The levels of sulfur and sulfate ions in the leaves of the plant were determined using elemental analysis and compared to the corresponding levels in comparable plant leaves from a less sulfate-rich environments. The analyses show the leaves from which we isolated blumenol C sulfate (1) to contain 35% more sulfur and 80% more sulfate than the other samples. Antimicrobial and antioxidant activities of compound 1 were tested against Escherichia coli, E. coli ampR and Bacillus subtilis as well as measured using complementary in vitro FRAP and ATBS assays, respectively. These bioactivities are comparable to those determined for structurally related megastigmanes. The sulfur and sulfate content of the plant leaves from the sulfate-rich environment was significantly higher than that of the other plants. Against this background of salt stress, we discuss a possible biosynthesis of blumenol C sulfate (1). Furthermore, there appears to be no benefit for the plant in terms of extended bioactivities. Hence, the formation of blumenol C sulfate (1) probably primarily serves the plant detoxification process

Structure of a highly phosphorylated lipopolysaccharide core in the ΔalgC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3)

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Bromination and Accompanying Rearrangement of the Polycyclic Oxetane 2,4-Oxytwistane

Bromination of the polycyclic oxetane 2,4-oxytwistane (<i>rac</i>-(1<i>R</i>,3<i>S</i>,4<i>R</i>,7<i>S</i>,9<i>R</i>,11<i>S</i>)-2-oxatetracyclo­[5.3.1.0^3,11.0^4,9]­undecane) was undertaken in order to form 2,4-dibromotwistane. The oxetane was subjected to the mild reagent combination CBr₄/Ph₃P in a fashion similar to that for the Appel and Corey–Fuchs reactions. NMR spectroscopy revealed that the isomeric dibromo compound 2,8-di­bro­mo­iso­twist­ane (2,8-dibromotricyclo­[4.3.1.0^3,7]­decane) was inadvertently formed. The conversion was prevented by migration of a C–C bond within the geometrically stressed C₁₀ framework. Computational chemistry was used to model the structure of the polycyclic oxetane and to assess the component of total ring strain energy due to the four-membered heterocycle. Mechanistic aspects behind the skeletal rearrangement are also discussed

Yellow Twig (<i>Nauclea</i> <i>orientalis</i>) from Thailand: Strictosamide as the Key Alkaloid of This Plant Species

Comprehensive phytochemical examination from different perspectives using preparative and analytical chromatographic techniques combined with spectroscopic/spectrometric methods of the so-called “yellow twig” Nauclea orientalis (L.) L. (Rubiaceae) led to the identification of 13 tryptamine-derived (=monoterpene-indole) alkaloids. The identified alkaloids comprise strictosamide and four of its glucosidic derivatives, three oxindole derivatives, and five yellow-colored angustine-type aglycones. Qualitative and quantitative HPLC analyses showed the enrichment of strictosamide in all studied organs. Based on these results, we performed metabolomic analyses of monoterpene-indole alkaloids and made a 1H NMR in vitro monitoring of enzymatic deglucosylation of strictosamide. A comparison of the stability of strictosamide and its enantiomer vincoside lactam by theoretical calculations was also performed revealing a slightly higher stability of vincoside lactam. Additionally, we conducted two different anti-feedant assays of strictosamide using larvae of the polyphageous moth Spodoptera littoralis Boisduval. The obtained results indicate that generally two different biosynthetic pathways are most likely responsible for the overall alkaloid composition in this plant. Strictosamide is the key compound in the broader pathway and most likely the source of the identified angustine-type aglycones, which may contribute significantly to the yellow color of the wood. Its cross-organ accumulation makes it likely that strictosamide is not only important as a reservoir for the further biosynthesis, but also acts in the plants’ defense strategy

MAPkinases regulate secondary metabolism, sexual development and light dependent cellulase regulation in Trichoderma reesei

Abstract The filamentous fungus Trichoderma reesei is a prolific producer of plant cell wall degrading enzymes, which are regulated in response to diverse environmental signals for optimal adaptation, but also produces a wide array of secondary metabolites. Available carbon source and light are the strongest cues currently known to impact secreted enzyme levels and an interplay with regulation of secondary metabolism became increasingly obvious in recent years. While cellulase regulation is already known to be modulated by different mitogen activated protein kinase (MAPK) pathways, the relevance of the light signal, which is transmitted by this pathway in other fungi as well, is still unknown in T. reesei as are interconnections to secondary metabolism and chemical communication under mating conditions. Here we show that MAPkinases differentially influence cellulase regulation in light and darkness and that the Hog1 homologue TMK3, but not TMK1 or TMK2 are required for the chemotropic response to glucose in T. reesei. Additionally, MAPkinases regulate production of specific secondary metabolites including trichodimerol and bisorbibutenolid, a bioactive compound with cytostatic effect on cancer cells and deterrent effect on larvae, under conditions facilitating mating, which reflects a defect in chemical communication. Strains lacking either of the MAPkinases become female sterile, indicating the conservation of the role of MAPkinases in sexual fertility also in T. reesei. In summary, our findings substantiate the previously detected interconnection of cellulase regulation with regulation of secondary metabolism as well as the involvement of MAPkinases in light dependent gene regulation of cellulase and secondary metabolite genes in fungi