RESUMO

Protein post-translational modification (PTM) is a ubiquitous process that occurs in most proteins. Lysine residues containing an ε-amino group are recognized as hotspots for the addition of different chemical groups. Lysine acetylation, extensively studied in histones, serves as an epigenetic hallmark capable of promoting changes in chromatin structure and availability. Acyl groups derived from molecules involved in carbohydrate and lipid metabolisms, such as lactate, succinate and hydroxybutyrate, were identified as lysine modifications of histones and other proteins. Lysine-acyltransferases do not exhibit significant substrate specificity concerning acyl donors. Furthermore, plant hormones harboring acyl groups often form conjugates with free amino acids to regulate their activity and function during plant physiological processes and responses, a process mediated by GH3 enzymes. Besides forming low-molecular weight conjugates, auxins have been shown to covalently modify proteins in bean seeds. Aside from auxins, other phytohormones with acyl groups are unexplored potential substrates for post-translational acylation of proteins. Using MS data searches, we revealed various proteins with lysine residues linked to auxin, abscisic acid, gibberellic acid, jasmonic acid, and salicylic acid. These findings raise compelling questions about the ability of plant hormones harboring carboxyl groups to serve as new candidates for protein acylation and acting in protein PTM and modulation.

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Saponins are amphipathic glycosides with detergent properties present in vegetables. These compounds, when ingested, can cause difficulties in absorbing nutrients from food and even induce inflammatory processes in the intestine. There is already some evidence that saponins can be degraded by ß-glucosidases of the GH3 family. In the present study, we evaluated, through computational tools, the possibility of a ß-glucosidase (AMBGL17) obtained from a metagenomic analysis of the Amazonian soil, to catalytically interact with a saponin present in soybean. For this, the amino acid sequence of AMBGL17 was used in a phylogenetic analysis to estimate its origin and to determine its three-dimensional structure. The 3D structure of the enzyme was used in a molecular docking analysis to evaluate its interaction with soy saponin as a ligand. The results of the phylogenetic analysis showed that AMBGL17 comes from a microorganism of the phylum Chloroflexi, probably related to species of the order Aggregatinales. Molecular docking showed that soybean saponin can interact with the catalytic site of AMBGL17, with the amino acid GLY345 being important in this catalytic interaction, especially with a ß-1,2 glycosidic bond present in the carbohydrate portion of saponin. In conclusion, AMBGL17 is an enzyme with interesting biotechnological potential in terms of mitigating the anti-nutritional and pro-inflammatory effects of saponins present in vegetables used for human and animal food.

Assuntos

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Auxin plays a central role in growth and plant development. To maintain auxin homeostasis, biological processes such as biosynthesis, transport, degradation, and reversible conjugation are essential. The Gretchen Hagen 3 (GH3) family genes codify for the enzymes that esterify indole-3-acetic acid (IAA) to various amino acids, which is a key process in the induction of somatic embryogenesis (SE). The GH3 family is one of the principal families of early response to auxin genes, exhibiting IAA-amido synthetase activity to maintain optimal levels of free auxin in the cell. In this study, we carried out a systematic identification of the GH3 gene family in the genome of Coffea canephora, determining a total of 18 CcGH3 genes. Analysis of the genetic structures and phylogenetic relationships of CcGH3 genes with GH3 genes from other plant species revealed that they could be clustered in two major categories with groups 1 and 2 of the GH3 family of Arabidopsis. We analyzed the transcriptome expression profiles of the 18 CcGH3 genes using RNA-Seq analysis-based data and qRT-PCR during the different points of somatic embryogenesis induction. Furthermore, the endogenous quantification of free and conjugated indole-3-acetic acid (IAA) suggests that the various members of the CcGH3 genes play a crucial role during the embryogenic process of C. canephora. Three-dimensional modeling of the selected CcGH3 proteins showed that they consist of two domains: an extensive N-terminal domain and a smaller C-terminal domain. All proteins analyzed in the present study shared a unique conserved structural topology. Additionally, we identified conserved regions that could function to bind nucleotides and specific amino acids for the conjugation of IAA during SE in C. canephora. These results provide a better understanding of the C. canephora GH3 gene family for further exploration and possible genetic manipulation.

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Prolactin (PRL) is a hormone principally secreted by lactotrophs of the anterior pituitary gland. Although the synthesis and exocytosis of this hormone are mainly under the regulation of hypothalamic dopamine (DA), the possibility that the anterior pituitary synthesises this catecholamine remains unclear. The present study aimed to determine if the anterior pituitary produces DA from the precursor l-3,4-dihydroxyphenylalanine (l-dopa). Accordingly, we investigated the expression of aromatic l-amino acid decarboxylase (AADC) enzyme and the transporter vesicular monoamine transporter 2 (VMAT2) in the anterior pituitary, AtT20 and GH3 cells by immunofluorescence and western blotting. Moreover, we investigated the production of DA from l-dopa and its release in vitro. Then, we explored the effects of l-dopa with respect to the secretion of PRL from anterior pituitary fragments. We observed that the anterior pituitary, AtT20 and GH3 cells express both AADC and VMAT2. Next, we detected an increase in DA content after anterior pituitary fragments were incubated with l-dopa. Also, the presence of l-dopa increased DA levels in incubation media and reduced PRL secretion. Likewise, the content of cellular DA increased after AtT20 cells were incubated with l-dopa. In addition, l-dopa reduced corticotrophin-releasing hormone-stimulated adrenocorticotrophic hormone release from these cells after AADC activity was inhibited by NSD-1015. Moreover, DA formation from l-dopa increased apoptosis and decreased proliferation. However, in the presence of NSD-1015, l-dopa decreased apoptosis and increased proliferation rates. These results suggest that the anterior pituitary synthesises DA from l-dopa by AADC and this catecholamine can be released from this gland contributing to the control of PRL secretion. In addition, our results suggest that l-dopa exerts direct actions independently from its metabolisation to DA.

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ß-Glucosidases are enzymes with high importance for many industrial processes, catalyzing the last and limiting step of the conversion of lignocellulosic material into fermentable sugars for biofuel production. However, ß-glucosidases are inhibited by high concentrations of the product (glucose), which limits the biofuel production on an industrial scale. For this reason, the structural mechanisms of tolerance to product inhibition have been the target of several studies. In this study, we performed in silico experiments, such as molecular dynamics (MD) simulations, free energy landscape (FEL) estimate, Poisson-Boltzmann surface area (PBSA), and grid inhomogeneous solvation theory (GIST) seeking a better understanding of the glucose tolerance and inhibition mechanisms of a representative GH1 ß-glucosidase and a GH3 one. Our results suggest that the hydrophobic residues Y180, W350, and F349, as well the polar one D238 act in a mechanism for glucose releasing, herein called "slingshot mechanism", dependent also on an allosteric channel (AC). In addition, water activity modulation and the protein loop motions suggest that GH1 ß-Glucosidases present an active site more adapted to glucose withdrawal than GH3, in consonance with the GH1s lower product inhibition. The results presented here provide directions on the understanding of the molecular mechanisms governing inhibition and tolerance to the product in ß-glucosidases and can be useful for the rational design of optimized enzymes for industrial interests.

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Beta-glucosidases are key enzymes involved in lignocellulosic biomass degradation for bioethanol production, which complete the final step during cellulose hydrolysis by converting cellobiose into glucose. Currently, industry requires enzymes with improved catalytic performance or tolerance to process-specific parameters. In this sense, metagenomics has become a powerful tool for accessing and exploring the biochemical biodiversity present in different natural environments. Here, we report the identification of a novel ß-glucosidase from metagenomic DNA isolated from soil samples enriched with decaying plant matter from a Secondary Atlantic Forest region. For this, we employed a functional screening approach using an optimized and synthetic broad host-range vector for library production. The novel ß-glucosidase - named Lfa2 - displays three GH3-family conserved domains and conserved catalytic amino acids D283 and E487. The purified enzyme was most active in pH 5.5 and at 50°C, and showed hydrolytic activity toward several pNP synthetic substrates containing ß-glucose, ß-galactose, ß-xylose, ß-fucose, and α-arabinopyranose, as well as toward cellobiose. Lfa2 showed considerable glucose tolerance, exhibiting an IC50 of 300 mM glucose and 30% of remaining activity in 600 mM glucose. In addition, Lfa2 retained full or slightly enhanced activity in the presence of several metal ions. Further, ß-glucosidase activity was increased by 1.7-fold in the presence of 10% (v/v) ethanol, a concentration that can be reached in conventional fermentation processes. Similarly, Lfa2 showed 1.7-fold enhanced activity at high concentrations of 5-hydroxymethyl furfural, one of the most important cellulase inhibitors in pretreated sugarcane bagasse hydrolysates. Moreover, the synergistic effect of Lfa2 on Bacillus subtilis GH5-CBM3 endoglucanase activity was demonstrated by the increased production of glucose (1.6-fold). Together, these results indicate that ß-glucosidase Lfa2 is a promissory enzyme candidate for utilization in diverse industrial applications, such as cellulosic biomass degradation or flavor enhancement in winemaking and grape processing.

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The ß-glucosidases (ß-D-glucoside glucohydrolase, EC 3.2.1.21) hydrolyze glycosidic bonds of alkyl-, amino-, or aryl-ß-D-glucosides, cyanogenic glucosides, disaccharides and short oligosaccharides and can also catalyze the synthesis of glycosyl-bonds between different molecules via transglycosylation. Due to their ubiquitous phylogenetic distribution, substrate diversity and ability to both hydrolyze and synthesize glycosidic bonds, the catalysis and regulation of ß-glucosidases have been extensively studied. Many ß-glucosidases are inhibited by the reaction product glucose, and reduced catalytic activity may limit the biotechnological and industrial applications of these enzymes and this has stimulated the search for ß-glucosidases that maintain their activity at high glucose concentrations. Studies of many glucose tolerant enzymes have been reported and due to the ongoing interest in these enzymes, here it has been reviewed this accumulated body of knowledge which provides valuable insights as to the kinetics, structure, regulation and evolution of glucose tolerant and glucose stimulated ß-glucosidases.

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Bifidobacterium is an important genus of probiotic bacteria colonizing the human gut. These bacteria can uptake oligosaccharides for the fermentative metabolism of hexoses and pentoses, producing lactate, acetate as well as short-chain fatty acids and propionate. These end-products are known to have important effects on human health. ß-glucosidases (EC 3.2.1.21) are pivotal enzymes for the metabolism and homeostasis of Bifidobacterium, since they hydrolyze small and soluble saccharides, typically producing glucose. Here we describe the cloning, expression, biochemical characterization and the first X-ray structure of a GH3 ß-glucosidase from the probiotic bacteria Bifidobacterium adolescentis (BaBgl3). The purified BaBgl3 showed a maximal activity at 45 °C and pH 6.5. Under the optimum conditions, BaBgl3 is highly active on 4-nitrophenyl-ß-d-glucopyranoside (pNPG) and, at a lesser degree, on 4-nitrophenyl-ß-d-xylopyranoside (pNPX, about 32% of the activity observed for pNPG). The 2.4 Šresolution crystal structure of BaBgl3 revealed a three-domain structure composed of a TIM barrel domain, which together with α/ß sandwich domain accommodate the active site and a third C-terminal fibronectin type III (FnIII) domain with unknown function. Modeling of the substrate in the active site indicates that an aspartate interacts with the hydroxyl group of the C6 present in pNPG but absent in pNPX, which explains the substrate preference. Finally, the enzyme is significantly stabilized by glycerol and galactose, resulting in considerable increase in the enzyme activity and its lifetime. The structural and biochemical studies presented here provide a deeper understanding of the molecular mechanisms of complex carbohydrates degradation utilized by probiotic bacteria as well as for the development of new prebiotic oligosaccharides.

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A phosphorolytic activity has been reported for beta-N-acetylglucosaminidases from glycoside hydrolase family 3 (GH3) giving an interesting explanation for an unusual histidine as catalytic acid/base residue and suggesting that members from this family may be phosphorylases [J. Biol. Chem. 2015, 290, 4887]. Here, we describe the characterization of Hsero1941, a GH3 beta-N-acetylglucosaminidase from the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae SmR1. The enzyme has significantly higher activity against pNP-beta-D-GlcNAcp (Km = 0.24 mM, kcat = 1.2 s-1, kcat/Km = 5.0 mM-1s-1) than pNP-beta-D-Glcp (Km = 33 mM, kcat = 3.3 × 10-3 s-1, kcat/Km = 9 × 10-4 mM-1s-1). The presence of phosphate failed to significantly modify the kinetic parameters of the reaction. The enzyme showed a broad aglycone site specificity, being able to hydrolyze sugar phosphates beta-D-GlcNAc 1P and beta-D-Glc 1P, albeit at a fraction of the rate of hydrolysis of aryl glycosides. GH3 beta-glucosidase EryBI, that does not have a histidine as the general acid/base residue, also hydrolyzed beta-D-Glc 1P, at comparable rates to Hsero1941. These data indicate that Hsero1941 functions primarily as a hydrolase and that phosphorolytic activity is likely adventitious. The prevalence of histidine as a general acid/base residue is not predictive, nor correlative, with GH3 beta-N-acetylglucosaminidases having phosphorolytic activity.

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The extracellular matrix (ECM) influences different physiological and pathophysiological aspects of the cell. The ECM consists in a complex network of macromolecules with characteristic biochemical properties that allow cells to sense their environments inducing different signals and changing cell behavior. The purpose of the present study was to evaluate the participation of different ECM proteins in cell morphology and its implication on motility, proliferation and hormone secretion in GH3 cells, a tumor pituitary cell. GH3 cells were cultured with a defined medium on collagens I/III and IV, fibronectin and laminin. GH3 cells express α2 integrin subunit de novo. The cells responded to the ECM proteins with differentiated cell surface morphologies and membrane protrusions. A rounded shape with small membrane blebs, weak substrate adhesion and high motility was observed in cells on C I/III and fibronectin, while on C IV and laminin cells were viewed elongated and adhered. Differences on actin cytoskeleton, cytoskeletal-associated vinculin and phospho-MLC showed that ECM proteins determine the cytoskeleton organization. Cell proliferation showed dependency on the ECM protein, observing a higher rate in cells on collagen I/III. Prolactin secretion was higher in cells with small blebs, but an unchangeable response to EGF was obtained with the ECM proteins, suggesting is a consequence of cortical actin arrangement. We ascribe the functional differences of the GH3 cells to the cytoskeletal organization. Overall, the data showed that ECM plays a critical role in GH3 cells modulating different cellular comportment and evidenced the importance of the ECM composition of pituitary adenomas.