Old Enzyme, New Role: The β-Glucosidase BglC of Streptomyces scabiei Interferes with the Plant Defense Mechanism by Hydrolyzing Scopolin

The beta-glucosidase BglC fulfills multiple functions in both primary metabolism and induction of pathogenicity of Streptomyces scabiei, the causative agent of common scab in root and tuber crops. Indeed, this enzyme hydrolyzes cellobiose and cellotriose to feed glycolysis with glucose directly and modifies the intracellular concentration of these cello-oligosaccharides, which are the virulence elicitors. The inactivation of bglC led to unexpected phenotypes such as the constitutive overproduction of thaxtomin A, the main virulence determinant of S. scabiei. In this work, we reveal a new target substrate of BglC, the phytoalexin scopolin. Removal of the glucose moiety of scopolin generates scopoletin, a potent inhibitor of thaxtomin A production. The hydrolysis of scopolin by BglC displayed substrate inhibition kinetics, which contrasts with the typical Michaelis–Menten saturation curve previously observed for the degradation of its natural substrate cellobiose. Our work, therefore, reveals that BglC targets both cello-oligosaccharide elicitors emanating from the hosts of S. scabiei, and the scopolin phytoalexin generated by the host defense mechanisms, thereby occupying a key position to fine-tune the production of the main virulence determinant thaxtomin A.

A beta-glucosidase of an insect herbivore determines both toxicity and deterrence of a dandelion defense metabolite

Gut enzymes can metabolize plant defense compounds and thereby affect the growth and fitness of insect herbivores. Whether these enzymes also influence feeding preference is largely unknown. We studied the metabolization of taraxinic acid β-D-glucopyranosyl ester (TA-G), a sesquiterpene lactone of the common dandelion (Taraxacum officinale) that deters its major root herbivore, the common cockchafer larva (Melolontha melolontha). We have demonstrated that TA-G is rapidly deglucosylated and conjugated to glutathione in the insect gut. A broad-spectrum M. melolontha β-glucosidase, Mm_bGlc17, is sufficient and necessary for TA-G deglucosylation. Using cross-species RNA interference, we have shown that Mm_bGlc17 reduces TA-G toxicity. Furthermore, Mm_bGlc17 is required for the preference of M. melolontha larvae for TA-G-deficient plants. Thus, herbivore metabolism modulates both the toxicity and deterrence of a plant defense compound. Our work illustrates the multifaceted roles of insect digestive enzymes as mediators of plant-herbivore interactions.

Structural and Catalytic Characterization of TsBGL, a β-Glucosidase From Thermofilum sp. ex4484_79

Beta-glucosidase is an enzyme that catalyzes the hydrolysis of the glycosidic bonds of cellobiose, resulting in the production of glucose, which is an important step for the effective utilization of cellulose. In the present study, a thermostable β-glucosidase was isolated and purified from the Thermoprotei Thermofilum sp. ex4484_79 and subjected to enzymatic and structural characterization. The purified β-glucosidase (TsBGL) exhibited maximum activity at 90°C and pH 5.0 and displayed maximum specific activity of 139.2μmol/min/mgzne against p-nitrophenyl β-D-glucopyranoside (pNPGlc) and 24.3μmol/min/mgzen against cellobiose. Furthermore, TsBGL exhibited a relatively high thermostability, retaining 84 and 47% of its activity after incubation at 85°C for 1.5h and 90°C for 1.5h, respectively. The crystal structure of TsBGL was resolved at a resolution of 2.14Å, which revealed a classical (α/β)8-barrel catalytic domain. A structural comparison of TsBGL with other homologous proteins revealed that its catalytic sites included Glu210 and Glu414. We provide the molecular structure of TsBGL and the possibility of improving its characteristics for potential applications in industries.

In silico analysis of promoter region and regulatory elements of glucan endo-1,3-beta-glucosidase encoding genes in Solanum tuberosum: cultivar DM 1-3 516 R44

Background Potato (Solanum tuberosum L.) is one of the most important food crops in the world. Pathogens remain as one of the major constraints limiting potato productivity. Thus, understanding of gene regulation mechanism of pathogenesis-related genes such as glucan endo-1,3-beta-glucosidase is a foundation for genetic engineering of potato for disease resistance and reduces the use of fungicides. In the present study, 19 genes were selected and attempts were made through in silico methods to identify and characterize the promoter regions, regulatory elements, and CpG islands of glucan endo-1,3-beta-glucosidase gene in Solanum tuberosum cultivar DM 1-3 516 R44. Results The current analysis revealed that single transcription start sites (TSSs) were present in 12/19 (63.2%) of promoter regions analyzed. The predictive score at a cutoff value of 0.8 for the majority (84.2%) of the promoter regions ranged from 0.90 to 1.00. The locations for 42% of the TSSs were below −500 bp relative to the start codon (ATG). MβGII was identified as the common promoter motif for 94.4% of the genes with an E value of 3.5e−001. The CpG analysis showed low CpG density in the promoter regions of most of the genes except for gene ID102593331 and ID: 102595860. The number of SSRs per gene ranged from 2 to 9 with repeat lengths of 2 to 6 bp. Evolutionary distances ranged from 0.685 to 0.770 (mean = 0.73), demonstrating narrower genetic diversity range. Phylogeny was inferred using the UPGMA method, and gene sequences from different species were found to be clustered together. Conclusion In silico identified regulatory elements in promoter regions will contribute to our understanding of the regulatory mechanism of glucan endo-1,3-beta-glucosidase genes and provide a promising target for genetic engineering to improve disease resistance in potatoes.

Effect of nitrogen sources and surfactants on cellulase production by fungal strains under submerged cultivation

Cellulase, as a complex enzyme containing endoglucanase, exoglucanase and beta‐glucosidase, acts to hydrolyze cellulose in order to be converted into glucose units. These subunits of cellulase synergistically hydrolyze the cellulosic substrate of industrial importance. The biosynthesis of celluase by microorganisms has been recently paid much attention Nitrogen is one of the important elements required for growth of microorganisms. Provision of utilizable form of nitrogen source to organisms is the basic requirement to be fulfilled for optimal growth. The use of surfactants in the production of hydrolytic enzymes is well known. Many reports have shown the stimulatory effects of surfactants on enzyme production by microorganisms in submerged or solid state fermentation. The effects of several surfactants on the activity of cellulase enzyme were also studied in this study. Among the nitrogen sources, proteose peptone and yeast extract were found quite effective nitrogen sources whereas tween -80 and oil of olive performed well as surfactants by fungal growth and production system.

Wild-type GBA1 increases the α-synuclein tetramer–monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice

Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson’s disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient–derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer–monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD–causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)–human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

Between two walls: Modeling the adsorption behavior of β-glucosidase A on bare and SAM-functionalised gold surfaces

The efficient immobilization of enzymes on surfaces remains a complex but central issue in the biomaterials field, which requires us to understand this process at the atomic level. Using a multi-scale approach combining all-atom molecular dynamics and coarse-grain Brownian dynamics simulations, we investigated the adsorption behavior of Beta-glucosidase A (BGA) on bare and SAM-functionalized gold surfaces. We monitored the enzyme position and orientation during the MD trajectories, and measured the contacts it forms with both surfaces. While the adsorption process has little impact on the protein conformation, it can nonetheless perturb its mechanical properties and catalytic activity. Our results show that compared to the SAM-functionalized surface, the adsorption of BGA on bare gold is more stable, but also less specific, and more likely to disrupt the enzyme's function. This observation emphasizes the fact that the structural organization of proteins at the solid interface is a keypoint when designing devices based on enzyme immobilization, as one must find an acceptable stability-activity trade-off.

A Label-free Optical Sensing Platform for Beta-glucosidase Activity Using Protein-inorganic Hybrid Nanoflowers

In this work, a convenient, label-free and dual-signal readout optical sensing platform for the sensitively and selectively determination of beta-glucosidase (β-Glu) activity was reported using protein-inorganic hybrid nanoflowers [BSA-Cu3(PO4)2·3H2O] possessing peroxidase-mimicking activity. The nanoflowers (NFs) were facilely synthesized through a self-assembled synthesis strategy at room temperature. The as-prepared NFs could catalytically convert the colorless and non-fluorescent Amplex Red into colored and highly fluorescent resorufin in the presence of hydrogen peroxide via electron transfer process. β-Glu could hydrolyze cyanogenic glycoside, using amygdalin (Amy) as a model, into cyanide ions (CN-), which can subsequently efficiently suppress the catalytic activity of NFs, accompanied with the fluorescence decrease and the color fading. The concentration of CN- was controlled by β-Glu-triggered enzymatic reaction of Amy. Thus, a sensing system was established for fluorescent and visual determination of β-Glu activity. Under the optimum conditions, the present fluorescent and visual bimodal sensing platform exhibited good sensitivity for β-Glu activity assay with a detection limit of 0.33 U·L-1. The sensing platform was further applied to determinate β-Glu in real samples and satisfactory results were attained. Additionally, the label-free optical sensing system can potentially be a promising candidate for β-Glu inhibitors screening.