A Unique β-1,2-Mannosyltransferase of Thermotoga maritima That Uses Di-myo-Inositol Phosphate as the Mannosyl Acceptor

In addition to di-myo-inositol-1,3′-phosphate (DIP), a compatible solute widespread in hyperthermophiles, the organic solute pool of Thermotoga maritima comprises 2-(O-β-d-mannosyl)-di-myo-inositol-1,3′-phosphate (MDIP) and 2-(O-β-d-mannosyl-1,2-O-β-d-mannosyl)-di-myo-inositol-1,3′-phosphate (MMDIP), two newly identified β-1,2-mannosides. In cells grown under heat stress, MDIP was the major solute, accounting for 43% of the total pool; MMDIP and DIP accumulated to similar levels, each corresponding to 11.5% of the total pool. The synthesis of MDIP involved the transfer of the mannosyl group from GDP-mannose to DIP in a single-step reaction catalyzed by MDIP synthase. This enzyme used MDIP as an acceptor of a second mannose residue, yielding the di-mannosylated compound. Minor amounts of the tri-mannosylated form were also detected. With a genomic approach, putative genes for MDIP synthase were identified in the genome of T. maritima, and the assignment was confirmed by functional expression in Escherichia coli. Genes with significant sequence identity were found only in the genomes of Thermotoga spp., Aquifex aeolicus, and Archaeoglobus profundus. MDIP synthase of T. maritima had maximal activity at 95°C and apparent K_m values of 16 mM and 0.7 mM for DIP and GDP-mannose, respectively. The stereochemistry of MDIP was characterized by isotopic labeling and nuclear magnetic resonance (NMR): DIP selectively labeled with carbon 13 at position C1 of the l-inositol moiety was synthesized and used as a substrate for MDIP synthase. This β-1,2-mannosyltransferase is unrelated to known glycosyltransferases, and within the domain Bacteria, it is restricted to members of the two deepest lineages, i.e., the Thermotogales and the Aquificales. To our knowledge, this is the first β-1,2-mannosyltransferase characterized thus far.Thermotoga maritima was first isolated from hot marine sediments on Vulcano Island, Italy, being able to grow between 55°C and 90°C (14). This strictly anaerobic bacterium ferments a variety of simple and complex carbohydrates to acetate, hydrogen, and CO₂ (10). In line with these metabolic traits, a substantial percentage of the genes annotated in the genome of this hyperthermophile are allocated to the metabolism of mono- and polysaccharides (8, 23). Therefore, T. maritima has been pointed out as a source of glycoside hydrolases with potential industrial relevance, namely, in processes of conversion of biomass into biofuels (3, 34).Like many other hyperthermophiles isolated from marine environments, Thermotoga maritima is slightly halophilic (optimum NaCl concentration of 2.7%, wt/vol) and has developed biochemical strategies to counterbalance the external osmotic pressure. The accumulation of low-molecular-mass organic compounds in the cytoplasm is the most common osmoadaptation mechanism, which enables a rapid response to fluctuations in the salinity of the external medium. Interestingly, the organic solutes encountered in organisms adapted to thrive in hot environments are clearly different from those used by mesophiles, leading to the view that osmolytes of (hyper)thermophiles could play an additional role as protectors of macromolecules and other cellular components against heat damage. This notion is further fuelled by the finding that the total pool of organic solutes of (hyper)thermophiles increases notably not only at supraoptimal salinity but also in response to heat stress conditions (30).Over the last decade, our team has directed considerable effort to assess the role of osmolytes in the thermo-adaptation strategies of hyperthermophiles. Despite the scarcity of genetic tools for manipulation of marine hyperthermophiles, a number of novel organic solutes were identified and the corresponding biosynthetic pathways characterized at the genetic and biochemical levels (15, 17, 30), providing critical knowledge for engaging in elucidation of the molecular basis of the whole process, from the sensing of stress to the synthesis of specific osmolytes. In this context, we recently reported the characterization of the pathway for synthesis of di-myo-inositol-1,3′-phosphate, the most common solute within hyperthermophiles (5). Additionally, the genes and enzymes involved in the relevant reaction steps were disclosed. The synthesis proceeds via a phosphorylated form of DIP, and the respective synthase is a membrane-associated enzyme that catalyzes the condensation of CDP-inositol with inositol-1-phosphate (26, 27).The solute pool in members of the order Thermotogales was investigated a few years ago (19). Thermotoga neapolitana responded to heat stress with a strong accumulation of DIP and DIP derivatives. One of the solutes was assigned to a mannosylated form of DIP, at that time designated di-mannosyl-di-myo-inositol phosphate; moreover, the presence of a second DIP derivative was proposed, but its structure remained elusive. Therefore, we set out to fully characterize the solute pool of Thermotoga spp. and to identify the genes and the enzyme(s) involved in the synthesis of the DIP derivatives. Members of the genus Thermotoga accumulated DIP and two mannosylated forms of this compound, herein fully characterized using isotopic labeling, NMR, and mass spectrometry. Moreover, the pathway for the synthesis of these novel solutes was identified, leading to the discovery of a unique β-1,2-mannosyltransferase that catalyzes the transfer of the mannosyl group from GDP-mannose to DIP.     

Laccase-catalyzed oxidation of phenolic compounds in organic media

Rhus vernificera laccase-catalyzed oxidation of phenolic compounds, i.e., (+)-catechin, (−)-epicatechin and catechol, was carried out in selected organic solvents to search for the favorable reaction medium. The investigation on reaction parameters showed that optimal laccase activity was obtained in hexane at 30 °C, pH 7.75 for the oxidation of (+)-catechin as well as for (−)-epicatechin, and in toluene at 35 °C, pH 7.25 for the oxidation of catechol. E_a and Q₁₀ values of the biocatalysis in the reaction media of the larger log p solvents like isooctane and hexane were relatively higher than those in the reaction media of lower log p solvents like toluene and dichloromethane. Maximum laccase activity in the organic media was found with 6.5% of buffer as co-solvent. A wider range of 0–28 μg protein/ml in hexane than that of 0–16.7 μg protein/ml in aqueous medium was observed for the linear increasing conversion of (+)-catechin. The kinetic studies revealed that in the presence of isooctane, hexane, toluene and dichloromethane, the K_m values were 0.77, 0.97, 0.53 and 2.9 mmol/L for the substrate of (+)-catechin; 0.43, 0.34, 0.14 and 3.4 mmol/L for (−)-epicatechin; 2.9, 1.8, 0.61 and 1.1 mmol/L for catechol, respectively, while the corresponding V_max values were 2.1 × 10⁻², 2.3 × 10⁻², 0.65 × 10⁻² and 0.71 × 10⁻² δA/μg protein min); 1.8 × 10⁻², 0.88 × 10⁻², 0.19 × 10⁻² and 1.0 × 10⁻² δA/μg protein min); 0.48 × 10⁻², 0.59 × 10⁻², 0.67 × 10⁻² and 0.54 × 10⁻² δA/μg protein min), respectively. FT-IR indicated the formation of probable dimer from (+)-catechin in organic solvent. These results suggest that this laccase has higher catalytic oxidation capacity of phenolic compounds in suitable organic media and favorite oligomers could be obtained.     

Structural Stability of Myoglobin in Organic Media

The structural stability of metmyoglobin in organic solvents and cosolvents was investigated aiming the choice of a suitable medium to perform its dissolution with maintenance of the native folding. The spectroscopic behavior of metmyoglobin solution in UV–Visible and circular dichroism was used to evaluate the solubility and the secondary structure. The results were dependable of the chemical structure of the organic compounds, their polarity and content, in the case of cosolvents. Protic solvents showed better ability than the aprotic ones for the biomolecule dissolution, since they are able to establish hydrogen bonds. Solvents with high polarity usually damage the secondary structure of the protein. Myoglobin was dissolved in pure methanol, ethylene glycol and glycerol. The secondary structure was retained in some extent. The controlled addition of sodium dodecyl sulfate to myoglobin aqueous solution changed the surface moiety of the protein. The complex was extracted to hexane with efficiency of 77%.     

Beta-nitrostyrene derivatives as potential antibacterial agents: a structure-property-activity relationship study

A multidisciplinary project was developed, combining the synthesis of a series of beta-nitrostyrene derivatives and the determination of their physicochemical parameters (redox potentials, partition coefficients), to the evaluation of the corresponding antibacterial activity. A complete conformational analysis was also performed, in order to get relevant structural information. Subsequently, a structure-property-activity (SPAR) approach was applied, through linear regression analysis, aiming at obtaining a putative correlation between the physicochemical parameters of the compounds investigated and their antibacterial activity (both against standard strains and clinical isolates). The beta-nitrostyrene compounds displayed a lower activity towards all the tested bacteria relative to the beta-methyl-beta-nitrostyrene analogues. This was observed particularly for the 3-hydroxy-4-methoxy-beta-methyl-beta-nitrostyrene (IVb) against the Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium). The SPAR results revealed the existence of a clear correlation between the redox potentials and the antibacterial activity of the series of beta-nitrostyrene derivatives under study.     

A rapid exocytosis mode in chromaffin cells with a neuronal phenotype

We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.     

l-Arginine supplementation improves rats’ antioxidant system and exercise performance

Reactive species have great importance in sports performance, once they can directly regulate energy production, muscular contraction, inflammation, and fatigue. Therefore, the redox control is essential for athletes’ performance. Studies demonstrated that l-arginine has an important role in the synthesis of urea, cell growth and production of nitric oxide, moreover, there are indications that it is also able to induce benefits to muscle antioxidant system through the upregulation of some antioxidant enzymes, and by inhibiting some pathways of reactive species production. Therefore, the aim of this study was to evaluate the effects of l-arginine supplementation on performance and oxidative stress of male rats (trained or not), submitted to a single session of high intensity exercise. Forty male Wistar rats were divided into four groups, control (C), control+l-arginine (C?+?A), trained (T), and trained+l-arginine (T?+?A). The aerobic training was conducted for 8 weeks. Data of maximum speed and time from tests were used as indicators of performance. Variables related to oxidative stress and antioxidant system were also evaluated. Aerobic training was capable to induce enhancements on animals’ exercise performance and on their redox state. Additionally, supplementation improved rats’ physical performance on both groups, control and trained. Different improvements between groups on the antioxidant capacity were observed. Nevertheless, considering the ergogenic effect of l-arginine and the lack of all positive adaptations promoted by the exercise training, untrained animals may be more exposed to oxidative damages after the practice of intense exercises.     

Crystal structure of Archaeoglobus fulgidus CTP:inositol-1-phosphate cytidylyltransferase, a key enzyme for di-myo-inositol-phosphate synthesis in (hyper)thermophiles

Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3′-phosphate-1′-phosphate from CDP-inositol and l-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent K_m values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the V_max determined at 90°C was 62.9 μmol · min⁻¹ · mg of protein⁻¹. The structure of IPCT is composed of a central seven-stranded mixed β-sheet, of which six β-strands are parallel, surrounded by six α-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X₄-P-K. CTP, l-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.