Handling Several Sugars at a Time: a Case Study of Xyloglucan Utilization by Ruminiclostridium cellulolyticum.

Xyloglucan utilization by Ruminiclostridium cellulolyticum was formerly shown to imply the uptake of large xylogluco-oligosaccharides, followed by cytosolic depolymerization into glucose, galactose, xylose, and cellobiose. This raises the question of how the anaerobic bacterium manages the simultaneous presence of multiple sugars. Using genetic and biochemical approaches targeting the corresponding metabolic pathways, we observed that, surprisingly, all sugars are catabolized, collectively, but glucose consumption is prioritized. Most selected enzymes display unusual features, especially the GTP-dependent hexokinase of glycolysis, which appeared reversible and crucial for xyloglucan utilization. In contrast, mutant strains lacking either galactokinase, cellobiose-phosphorylase, or xylulokinase still catabolize xyloglucan but display variably altered growth. Furthermore, the xylogluco-oligosaccharide depolymerization process appeared connected to the downstream pathways through an intricate network of competitive and noncompetitive inhibitions. Altogether, our data indicate that xyloglucan utilization by R. cellulolyticum relies on an energy-saving central carbon metabolism deviating from current bacterial models, which efficiently prevents carbon overflow. IMPORTANCE The study of the decomposition of recalcitrant plant biomass is of great interest as the limiting step of terrestrial carbon cycle and to produce plant-derived valuable chemicals and energy. While extracellular cellulose degradation and catabolism have been studied in detail, few publications describe the complete metabolism of hemicelluloses and, to date, the published models are limited to the extracellular degradation and sequential entry of simple sugars. Here, we describe how the model anaerobic bacterium Ruminiclostridium cellulolyticum deals with the synchronous intracellular release of glucose, galactose, xylose, and cellobiose upon cytosolic depolymerization of imported xyloglucan oligosaccharides. The described novel metabolic strategy involves the simultaneous activity of different metabolic pathways coupled to a network of inhibitions controlling the carbon flux and is distinct from the ubiquitously observed sequential uptake and metabolism of carbohydrates known as the diauxic shift. Our results highlight the diversity of cellular responses related to a complex environment.

Synthesis, biological evaluation and modeling of hybrids from tetrahydro-1H-pyrazolo[3,4-b]quinolines as dual cholinestrase and COX-2 inhibitors.

New tetrahydro-1H-pyrazolo[3,4-b]quinoline derivatives were designed, synthesized and characterized as dual anticholinestrase and cyclooxygenase-2 inhibitors. The in vitro and in vivo anti-cholinesterase evaluation exhibited promising activities with lower hepatotoxicity for many candidates compared to tacrine as a reference. Furthermore, their anti-inflammatory activity using in vitro (COX-1/COX-2) inhibitory assay demonstrated superior activity to celecoxib with higher selectivity indices for some compounds. In addition, some candidates showed extended anti-inflammatory activity by inhibiting COX-2 protein induction. Besides, in silico docking experiments of the active compounds against hAChE rationalized the observed in vitro AChE inhibitory activity. In conclusion, this work provides an extension of the chemical space of tetrahydro-1H-pyrazolo[3,4-b]quinoline chemotype for the anticholinestrase and anti-inflammatory activity. This would aid to minimize the possible neuroinflammation linked to the pathogenesis of Alzheimer's disease.

The xyl-doc gene cluster of Ruminiclostridium cellulolyticum encodes GH43- and GH62-α-l-arabinofuranosidases with complementary modes of action.

BACKGROUND: The α-l-arabinofuranosidases (α-l-ABFs) are exoenzymes involved in the hydrolysis of α-l-arabinosyl linkages in plant cell wall polysaccharides. They play a crucial role in the degradation of arabinoxylan and arabinan and they are used in many biotechnological applications. Analysis of the genome of R. cellulolyticum showed that putative cellulosomal α-l-ABFs are exclusively encoded by the xyl-doc gene cluster, a large 32-kb gene cluster. Indeed, among the 14 Xyl-Doc enzymes encoded by this gene cluster, 6 are predicted to be α-l-ABFs belonging to the CAZyme families GH43 and GH62. RESULTS: The biochemical characterization of these six Xyl-Doc enzymes revealed that four of them are α-l-ABFs. GH4316-1229 (RcAbf43A) which belongs to the subfamily 16 of the GH43, encoded by the gene at locus Ccel_1229, has a low specific activity on natural substrates and can cleave off arabinose decorations located at arabinoxylan chain extremities. GH4310-1233 (RcAbf43Ad2,3), the product of the gene at locus Ccel_1233, belonging to subfamily 10 of the GH43, can convert the double arabinose decorations present on arabinoxylan into single O2- or O3-linked decorations with high velocity (k cat = 16.6 ± 0.6 s-1). This enzyme acts in synergy with GH62-1234 (RcAbf62Am2,3), the product of the gene at locus Ccel_1234, a GH62 α-l-ABF which hydrolyzes α-(1 → 3) or α-(1 → 2)-arabinosyl linkages present on polysaccharides and arabinoxylooligosaccharides monodecorated. Finally, a bifunctional enzyme, GH62-CE6-1240 (RcAbf62Bm2,3Axe6), encoded by the gene at locus Ccel_1240, which contains a GH62-α-l-ABF module and a carbohydrate esterase (CE6) module, catalyzes deacylation of plant cell wall polymers and cleavage of arabinosyl mono-substitutions. These enzymes are also active on arabinan, a component of the type I rhamnogalacturonan, showing their involvement in pectin degradation. CONCLUSION: Arabinofuranosyl decorations on arabinoxylan and pectin strongly inhibit the action of xylan-degrading enzymes and pectinases. α-l-ABFs encoded by the xyl-doc gene cluster of R. cellulolyticum can remove all the decorations present in the backbone of arabinoxylan and arabinan, act synergistically, and, thus, play a crucial role in the degradation of plant cell wall polysaccharides.

Synthesis of some new amide-linked bipyrazoles and their evaluation as anti-inflammatory and analgesic agents.

Four series of new bipyrazoles comprising the N-phenylpyrazole scaffold linked to polysubstituted pyrazoles or to antipyrine moiety through different amide linkages were synthesized. The synthesized compounds were evaluated for their anti-inflammatory and analgesic activities. In vitro COX-1/COX-2 inhibition study revealed that compound 16b possessed the lowest IC50 value against both COX-1 and COX-2. Moreover, the effect of the most promising compounds on inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) protein expression in lipopolysaccharide (LPS)-activated rat monocytes was also investigated. The results revealed that some of the synthesized compounds showed anti-inflammatory and/or analgesic activity with less ulcerogenic potential than the reference drug diclofenac sodium and are well tolerated by experimental animals. Moreover, they significantly inhibited iNOS and COX-2 protein expression induced by LPS stimulation. Compounds 16b and 18 were proved to display anti-inflammatory activity superior to diclofenac sodium and analgesic activity equivalent to it with minimal ulcerogenic potential.