SAXS Reveals the Stabilization Effects of Modified Sugars on Model Proteins.

Many proteins are usually not stable under different stresses, such as temperature and pH variations, mechanical stresses, high concentrations, and high saline contents, and their transport is always difficult, because they need to be maintained in a cold regime, which is costly and very challenging to achieve in remote areas of the world. For this reason, it is extremely important to find stabilizing agents that are able to preserve and protect proteins against denaturation. In the present work, we investigate, by extensively using synchrotron small-angle X-ray scattering experiments, the stabilization effect of five different sugar-derived compounds developed at ExtremoChem on two model proteins: myoglobin and insulin. The data analysis, based on a novel method that combines structural and thermodynamic features, has provided details about the physical-chemical processes that regulate the stability of these proteins in the presence of stabilizing compounds. The results clearly show that some modified sugars exert a greater stabilizing effect than others, being able to maintain the active forms of proteins at temperatures higher than those in which proteins, in the absence of stabilizers, reach denatured states.

The structural characterization of a glucosylglycerate hydrolase provides insights into the molecular mechanism of mycobacterial recovery from nitrogen starvation.

Bacteria are challenged to adapt to environmental variations in order to survive. Under nutritional stress, several bacteria are able to slow down their metabolism into a nonreplicating state and wait for favourable conditions. It is almost universal that bacteria accumulate carbon stores to survive during this nonreplicating state and to fuel rapid proliferation when the growth-limiting stress disappears. Mycobacteria are exceedingly successful in their ability to become dormant under harsh circumstances and to be able to resume growth when conditions are favourable. Rapidly growing mycobacteria accumulate glucosylglycerate under nitrogen-limiting conditions and quickly mobilize it when nitrogen availability is restored. The depletion of intracellular glucosyl-glycerate levels in Mycolicibacterium hassiacum (basonym Mycobacterium hassiacum) was associated with the up-regulation of the gene coding for glucosylglycerate hydrolase (GgH), an enzyme that is able to hydrolyse glucosylglycerate to glycerate and glucose, a source of readily available energy. Highly conserved among unrelated phyla, GgH is likely to be involved in bacterial reactivation following nitrogen starvation, which in addition to other factors driving mycobacterial recovery may also provide an opportunity for therapeutic intervention, especially in the serious infections caused by some emerging opportunistic pathogens of this group, such as Mycobacteroides abscessus (basonym Mycobacterium abscessus). Using a combination of biochemical methods and hybrid structural approaches, the oligomeric organization of M. hassiacum GgH was determined and molecular determinants of its substrate binding and specificity were unveiled.

The effect of new compounds in stabilizing downstream monoclonal antibody (mAb) process intermediates.

Determining the stability of downstream process (DSP) intermediates is an extremely important parameter used to maintain product quality attributes within their acceptance ranges. The IgG4 monoclonal antibody studied (mAb1) showed aggregation under acidic conditions, inhibiting the use of low pH treatment to inactivate endogenous retroviruses, and poor virus filtration performance. Both manufacturing steps are included in mAb DSP for viral clearance. The impact of several new compounds on the aggregation and stabilization of mAb1 in process intermediate pools encountered during these critical DSP steps was investigated. Results showed that, in the presence of a protein stabilizer at pH 3.2, 27% less aggregation was observed compared to controls, during the low pH treatment for viral inactivation. The impact of a novel protein stabilizer on virus filter throughput during mAb1 filtration was compared to L-arginine using an innovative high-throughput automation technique. Compared to control experiments without additives, conditions were found where a 70% increase in filter volumetric throughput was achieved in the presence of the novel stabilizer, and a 56% decrease in volumetric throughput observed with L-arginine. These findings present the possibility of using these novel compounds to stabilize proteins during DSP and permitting the use of platform DSP elements such as low pH treatment and high-throughput virus filtration to challenging and unstable proteins.

Syntheses of the plant auxin conjugate 2-O-(indole-3-acetyl)-myo-inositol IAInos.

The plant hormone conjugate 2-O-(indole-3-acetyl)-myo-inositol (IAInos) has been selectively prepared for the first time by two routes from myo-inositol. One of the syntheses depended upon the construction of the 3-indoleacetyl group by a Fischer indole synthesis on an unreactive axial hydroxyl group, while the other via a direct acylation of the equatorially orientated hydroxy group created by conformational constraint of the cyclohexane ring. The latter synthesis produced IAInos in 5 steps and 29% overall yield.

Host glycan sugar-specific pathways in Streptococcus pneumoniae: galactose as a key sugar in colonisation and infection [corrected].

The human pathogen Streptococcus pneumoniae is a strictly fermentative organism that relies on glycolytic metabolism to obtain energy. In the human nasopharynx S. pneumoniae encounters glycoconjugates composed of a variety of monosaccharides, which can potentially be used as nutrients once depolymerized by glycosidases. Therefore, it is reasonable to hypothesise that the pneumococcus would rely on these glycan-derived sugars to grow. Here, we identified the sugar-specific catabolic pathways used by S. pneumoniae during growth on mucin. Transcriptome analysis of cells grown on mucin showed specific upregulation of genes likely to be involved in deglycosylation, transport and catabolism of galactose, mannose and N acetylglucosamine. In contrast to growth on mannose and N-acetylglucosamine, S. pneumoniae grown on galactose re-route their metabolic pathway from homolactic fermentation to a truly mixed acid fermentation regime. By measuring intracellular metabolites, enzymatic activities and mutant analysis, we provide an accurate map of the biochemical pathways for galactose, mannose and N-acetylglucosamine catabolism in S. pneumoniae. Intranasal mouse infection models of pneumococcal colonisation and disease showed that only mutants in galactose catabolic genes were attenuated. Our data pinpoint galactose as a key nutrient for growth in the respiratory tract and highlights the importance of central carbon metabolism for pneumococcal pathogenesis.

A unique glyceryl diglycoside identified in the thermophilic, radiation-resistant bacterium Rubrobacter xylanophilus.

The solute pool of the actinobacterium Rubrobacter xylanophilus has been investigated as a function of the growth temperature and concentration of NaCl in the medium (Empadinhas et al. Extremophiles 11: 667-673, 2007). Changing the carbon source from glucose to maltose in a minimal growth medium led to the accumulation of an unknown organic compound whose structure was investigated by NMR and confirmed by chemical synthesis in the present study as: (2R)-2-(1-O-α-D-mannopyranosyl)-3-(1-O-α-D-glucopyranosyl)-D-glycerate (MGlyG). In addition to this newly identified diglycoside, the solute pool of R. xylanophilus included trehalose, mannosylglycerate, di-myo-inositol phosphate and di-N-acetyl-glucosamine phosphate. The structure of MGlyG was established by NMR and confirmed by chemical synthesis. The availability of g-amounts of the synthetic material allowed us to perform stabilization tests on three model enzymes (malate dehydrogenase, staphylococcal nuclease, and lysozyme), and compare the efficacy of MGlyG with other natural glyceryl glycosides, such as α-D-mannosyl-D-glycerate, α-D-glucosyl-D-glycerate and α-D-glucosyl-(1 → 6)-α-D-glucosyl-(1 → 2)-D-glycerate.

Mycobacterium hassiacum recovers from nitrogen starvation with up-regulation of a novel glucosylglycerate hydrolase and depletion of the accumulated glucosylglycerate.

Some microorganisms accumulate glucosylglycerate (GG) during growth under nitrogen deprivation. However, the molecular mechanisms underlying the role of GG and the regulation of its levels in the nitrogen stress response are elusive. Since GG is required for biosynthesis of mycobacterial methylglucose lipopolysaccharides (MGLP) we examined the molecular mechanisms linking replenishment of assimilable nitrogen to nitrogen-starved M. hassiacum with depletion of GG accumulated during nitrogen deficiency. To probe the involvement of a newly identified glycoside hydrolase in GG depletion, we produced the mycobacterial enzyme recombinantly and confirmed the specific hydrolysis of GG (GG hydrolase, GgH) in vitro. We have also observed a pronounced up-regulation of GgH mRNA in response to the nitrogen shock, which positively correlates with GG depletion in vivo and growth stimulation, implicating GgH in the recovery process. Since GgH orthologs seem to be absent from most slowly-growing mycobacteria including M. tuberculosis, the disclosure of the GgH function allows reconfiguration of the MGLP pathway in rapidly-growing species and accommodation of this possible regulatory step. This new link between GG metabolism, MGLP biosynthesis and recovery from nitrogen stress furthers our knowledge on the mycobacterial strategies to endure a frequent stress faced in some environments and during long-term infection.

The synthesis of compatible solute analogues-solvent effects on selective glycosylation.

Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-d-thioglucoside and ethyl 6-O-acetyl-2,3,4-tribenzyl-1-d-thiogalactoside, as a mixture of anomers, were employed in the study of the influence of solvent in the stereoselectivity of the glycosylation reaction with small and reactive acceptors. High α-selectivities were obtained in the glycosylation reactions using NIS/TfOH as activator and ethyl ether as the solvent at -60°C. Other solvent mixtures such as dichloromethane, THF, THF/ethyl ether and toluene/dioxane were not nearly as selective. The corresponding thiogalactoside underwent similar glycosylations with the same solvents but with low anomer selectivity. These glycosides are key intermediates for the synthesis of new analogues of compatible solutes.

Synthesis of potassium (2R)-2-O-alpha-d-glucopyranosyl-(1-->6)-alpha-d-glucopyranosyl-2,3-dihydroxypropanoate a natural compatible solute.

Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-thio-d-glucopyranoside, as a mixture of anomers, was employed for the stereoselective synthesis of the potassium salt of (2R)-2-O-alpha-d-glucopyranosyl-(1-->6)-alpha-d-glucopyranosyl-2,3-dihydroxypropanoic acid (alpha-d-glucosyl-(1-->6)-alpha-d-glucosyl-(1-->2)-d-glyceric acid, GGG), a recently isolated compatible solute. The alpha-anomer was by far the major product of both glycosylation reactions using NIS/TfOH as activator.