Chemical profiling of the deacetylase activity of acetyl xylan esterase A (AxeA) variants on chitooligosaccharides using hydrophilic interaction chromatography-mass spectrometry

Chitosan oligosaccharides (oligomers of (GlcNAc)_x(GlcN)_y) are used in the pharmaceutical, cosmetic and food industries and are reported to have therapeutic benefits. However, it is unknown whether their biological activity depends on the degree of deacetylation or the sequence of residues within the oligomer. We report here the development of a random mutagenesis method for directed evolution of Streptomyces lividans acetyl xylan esterase (AxeA), which we previously showed is able to deacetylate chitinous substrate, in order to obtain chitooligosaccharides with well-defined structural properties. A colorimetric assay was used to pre-screen libraries for p-nitrophenol acetate hydrolysis activity and an HPLC-UV absorbance assay was optimized to subsequently screen for deacetylase activity toward hexa-N-acetyl-glucosamine substrate (GlcNAc)₆. Native AxeA and two variants displaying > 50% deacetylation of the oligohexamer substrate after reaction at 50 °C for 24 h in diluted culture supernatant were then selected for detailed analysis of the enzymatic products. A HILIC (hydrophilic interaction chromatography)-mode LC method was developed for profiling the deacetylated chitooligosaccharide products and HILIC-MS/MS sequencing revealed that ca. 30 different deacetylation products ranging from (GlcNAc)₅(GlcN)₁ to (GlcNAc)₁(GlcN)₅ and isomers thereof were produced. The AxeA variants produced, on average, 26% more unique products than the native enzyme; however, none were able to fully deacetylate the substrate to make (GlcN)₆. The long term goal of this multidisciplinary approach is to improve the activity of chitosan oligosaccharides to an industrially applicable level.     

Drinking and driving pancreatitis: links between endoplasmic reticulum stress and autophagy

Alcohol abuse is the leading etiologic factor of pancreatitis, although many heavy drinkers do not develop pancreatic damage. Alcohol promotes pancreatitis through a combination of remote (e.g., increased gut permeability to bacterial products such as lipopolysaccharide) and more proximal effects (e.g., altered pancreatic cholinergic inputs), including oxidative damage at the level of the pancreatic acinar cell. Recent evidence indicates that alcohol exposure to rodents disturbs proteostasis in the exocrine pancreas, an effect counterbalanced by homeostatic processes that include both the unfolded protein response (UPR) and autophagy. A corollary to this notion is that pancreatitis results when adaptive responses are insufficiently robust to alleviate the cellular stress caused by alcohol.     

Ferulic acid dehydrodimers from wheat bran: isolation,purification and antioxidant properties of 8-0-4-diferulic acid

Summary

Wheat bran contains several ester-linked dehydrodimers of ferulic acid, which were detected and quantified after sequential alkaline hydrolysis. The major dimers released were: trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (5–8-BendiFA), (Z)-β-(4-[(E)-2-carboxyvinyl]-2-methoxy-phenoxy)-4-hydroxy-3-methoxycinnamic acid (8-O-4-diFA) and (E,E)-4,4′-dihydroxy-5,5′-dimethoxy-3,3′-bicinnamic acid (5–5-diFA). trans-7-hydroxy-1-(4-hydroxy-3methoxyphenyl)-6-methoxy-1,2-dihydro-naphthalene-2,3-dicarboxylic acid (8–8-diFA cyclic form) and 4,4′-dihydroxy-3,3′-dimethoxy-β,β'-bicinnamic acid (8–8-diFA non cyclic form) were not detected. One of the most abundant dimers, 8-O-4-diFA, was purified from de-starched wheat bran after alkaline hydrolysis and preparative HPLC. The resultant product was identical to the chemically synthesised 8-O-4-dimer by TLC and HPLC as confirmed by ¹H-NMR and mass spectrometry. The absorption maxima and absorption coefficients for the synthetic compound in ethanol were: λ_max: 323 nm, λ_min: 258 nm, ε_λmax (M^?1cm^?1): 24800 ± 2100 and ε₂₈₀ (M^?1cm^?1): 19700 ± 1100. The antioxidant properties of 8-O-4-diFA were assessed using: (a) inhibition of ascorbate/iron-induced peroxidation of phosphatidylcholine liposomes and; (b) scavenging of the radical cation of 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulphonate) (ABTS) relative to the water-soluble vitamin E analogue, Trolox C. The 8-O-4-diFA was a better antioxidant than ferulic acid in both lipid and aqueous phases. This is the first report of the antioxidant activity of a natural diferulate obtained from a plant.     

Using Multiscale Spatial Models to Assess Potential Surrogate Habitat for an Imperiled Reptile

In evaluating conservation and management options for species, practitioners might consider surrogate habitats at multiple scales when estimating available habitat or modeling species’ potential distributions based on suitable habitats, especially when native environments are rare. Species’ dependence on surrogates likely increases as optimal habitat is degraded and lost due to anthropogenic landscape change, and thus surrogate habitats may be vital for an imperiled species’ survival in highly modified landscapes. We used spatial habitat models to examine a potential surrogate habitat for an imperiled ambush predator (eastern diamondback rattlesnake, Crotalus adamanteus; EDB) at two scales. The EDB is an apex predator indigenous to imperiled longleaf pine ecosystems (Pinus palustris) of the southeastern United States. Loss of native open-canopy pine savannas and woodlands has been suggested as the principal cause of the species’ extensive decline. We examined EDB habitat selection in the Coastal Plain tidewater region to evaluate the role of marsh as a potential surrogate habitat and to further quantify the species’ habitat requirements at two scales: home range (HR) and within the home range (WHR). We studied EDBs using radiotelemetry and employed an information-theoretic approach and logistic regression to model habitat selection as use vs. availability. We failed to detect a positive association with marsh as a surrogate habitat at the HR scale; rather, EDBs exhibited significantly negative associations with all landscape patches except pine savanna. Within home range selection was characterized by a negative association with forest and a positive association with ground cover, which suggests that EDBs may use surrogate habitats of similar structure, including marsh, within their home ranges. While our HR analysis did not support tidal marsh as a surrogate habitat, marsh may still provide resources for EDBs at smaller scales.     

How does climate change cause extinction?

Anthropogenic climate change is predicted to be a major cause of species extinctions in the next 100 years. But what will actually cause these extinctions? For example, will it be limited physiological tolerance to high temperatures, changing biotic interactions or other factors? Here, we systematically review the proximate causes of climate-change related extinctions and their empirical support. We find 136 case studies of climatic impacts that are potentially relevant to this topic. However, only seven identified proximate causes of demonstrated local extinctions due to anthropogenic climate change. Among these seven studies, the proximate causes vary widely. Surprisingly, none show a straightforward relationship between local extinction and limited tolerances to high temperature. Instead, many studies implicate species interactions as an important proximate cause, especially decreases in food availability. We find very similar patterns in studies showing decreases in abundance associated with climate change, and in those studies showing impacts of climatic oscillations. Collectively, these results highlight our disturbingly limited knowledge of this crucial issue but also support the idea that changing species interactions are an important cause of documented population declines and extinctions related to climate change. Finally, we briefly outline general research strategies for identifying these proximate causes in future studies.     

Molecular basis for nucleotide-binding specificity: role of the exocyclic amino group "N2" in recognition by a guanylyl-ribonuclease

Proteins interact with nucleotides to perform a multitude of functions within cells. These interactions are highly specific; however, the molecular basis for this specificity is not well understood. To identify factors critical for protein-guanine nucleotide recognition the binding of two closely related ligands, guanosine 3'-monophosphate (3'GMP) and inosine 3'-monophosphate (3'IMP), to Ribonuclease Sa (RNase Sa), a small, guanylyl-endoribonuclease from Streptomyces aureofaciens, was compared using isothermal titration calorimetry, NMR, X-ray crystallography and molecular dynamics simulations. This comparison has allowed for the determination of the contribution of the exocyclic amino group "N2" of the guanine base to nucleotide binding specificity. Calorimetric measurements indicate that RNase Sa has a higher affinity for 3'GMP (K=(1.5+/-0.2)x10(5)) over 3'IMP (K=(3.1+/-0.2)x10(4)) emphasizing the importance of N2 as a key determinant of RNase Sa guanine binding specificity. This result was unexpected as the published structural data for RNase Sa in complex with 3'GMP showed only a potential long-range interaction (>3.3A) between N2 and the side-chain of Glu41 of RNase Sa. The observed difference in affinity is largely due to a reduction in the favorable enthalpy change by 10 kJ/mol for 3'IMP binding as compared to 3'GMP that is accompanied by a significant difference in the heat capacity changes observed for binding the two ligands. To aid interpretation of the calorimetric data, the first crystal structure of a small, guanylyl ribonuclease bound to 3'IMP was determined to 2.0 A resolution. This structure has revealed small yet unexpected changes in the ligand conformation and differences in the conformations of the side-chains contacting the sugar and phosphate moieties as compared to the 3'GMP complex. The structural data suggest the less favorable enthalpy change is due to an overall lengthening of the contacts between RNase Sa and 3'IMP as compared to 3'GMP. The long-range interaction between N2 and Glu41 is critical for positioning of the nucleotide in the binding cleft for optimal contact formation. Thus, combined, the data demonstrate how a long-range interaction can have a significant impact on nucleotide binding affinity and energetics.